Rage fusion proteins and methods of use

ABSTRACT

Disclosed are RAGE fusion proteins comprising RAGE polypeptide sequences linked to a second, non-RAGE polypeptide. The RAGE fusion protein may utilize a RAGE polypeptide domain comprising a RAGE ligand binding site and an interdomain linker directly linked to an immunoglobulin C H 2 domain. Such fusion proteins may provide specific, high affinity binding to RAGE ligands. Also disclosed is the use of the RAGE fusion proteins as therapeutics for RAGE-mediated pathologies.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application claims priority under 35 USC 119(e) from U.S.Provisional Patent Application Ser. No. 60/598,555 filed Aug. 3, 2004.The disclosure of U.S. Provisional Patent Application 60/598,555 ishereby incorporated by reference in its entirety herein.

FIELD OF THE INVENTION

The present invention relates to regulation of the Receptor for AdvancedGlycated End products (RAGE). More particularly, the present inventiondescribes fusion proteins comprising a RAGE polypeptide, methods ofmaking such fusion proteins, and the use of such proteins for treatmentof RAGE-based disorders.

BACKGROUND

Incubation of proteins or lipids with aldose sugars results innonenzymatic glycation and oxidation of amino groups on proteins to formAmadori adducts. Over time, the adducts undergo additionalrearrangements, dehydrations, and cross-linking with other proteins toform complexes known as Advanced Glycosylation End Products (AGEs).Factors which promote formation of AGEs include delayed protein turnover(e.g. as in amyloidoses), accumulation of macromolecules having highlysine content, and high blood glucose levels (e.g. as in diabetes)(Hori et al., J. Biol. Chem. 270: 25752-761, (1995)). AGEs have beenimplicated in a variety of disorders including complications associatedwith diabetes and normal aging.

AGEs display specific and saturable binding to cell surface receptors onmonocytes, macrophages, endothelial cells of the microvasculature,smooth muscle cells, mesengial cells, and neurons. The Receptor forAdvanced Glycated Endproducts (RAGE) is a member of the immunoglobulinsupergene family of molecules. The extracellular (N-terminal) domain ofRAGE includes three immunoglobulin-type regions: one V (variable) typedomain followed by two C-type (constant) domains (Neeper et al., J.Biol. Chem., 267:14998-15004 (1992); Schmidt et al., Circ. (Suppl.)96#194 (1997)). A single transmembrane spanning domain and a short,highly charged cytosolic tail follow the extracellular domain. TheN-terminal, extracellular domain can be isolated by proteolysis of RAGEor by molecular biological approaches to generate soluble RAGE (sRAGE)comprised of the V and C domains.

RAGE is expressed on multiple cell types including leukocytes, neurons,microglial cells and vascular endothelium (e.g., Hori et al., J. Biol.Chem., 270:25752-761 (1995)). Increased levels of RAGE are also found inaging tissues (Schleicher et al., J. Clin. Invest., 99 (3): 457-468(1997)), and the diabetic retina, vasculature and kidney (Schmidt etal., Nature Med., 1:1002-1004 (1995)).

In addition to AGEs, other compounds can bind to and modulate RAGE. RAGEbinds to multiple functionally and structurally diverse ligandsincluding amyloid beta (AP), serum amyloid A (SAA), Advanced GlycationEnd products (AGEs), S100 (a proinflammatory member of the Calgranulinfamily), carboxymethyl lysine (CML), amphoterin and CD11b/CD18(Bucciarelli et al., Cell Mol. Life Sci., 59:1117-128 (2002); Chavakiset al., Microbes Infect., 6:1219-1225 (2004); Kokkola et al., Scand. J.Immunol., 61:1-9 (2005); Schmidt et al., J. Clin. Invest., 108:949-955(2001); Rocken et al., Am. J. Pathol., 162:1213-1220 (2003)).

Binding of ligands such as AGEs, S100/calgranulin, β-amyloid, CML(N^(ε)-Carboxymethyl lysine), and amphoterin to RAGE has been shown tomodify expression of a variety of genes. These interactions may theninitiate signal transduction mechanisms including p38 activation,p21ras, MAP kinases, Erk1-2 phosphorylation, and the activation of thetranscriptional mediator of inflammatory signaling, NF-κB (Yeh et al.,Diabetes, 50:1495-1504 (2001)). For example, in many cell types,interaction between RAGE and its ligands can generate oxidative stress,which thereby results in activation of the free radical sensitivetranscription factor NF-κB, and the activation of NF-κB regulated genes,such as the cytokines IL-1β and TNF-α. Furthermore, RAGE expression isupregulated via NF-κB and shows increased expression at sites ofinflammation or oxidative stress (Tanaka et al., J. Biol. Chem.,275:25781-25790 (2000)). Thus, an ascending and often detrimental spiralmay be fueled by a positive feedback loop initiated by ligand binding.

Activation of RAGE in different tissues and organs can lead to a numberof pathophysiological consequences. RAGE has been implicated in avariety of conditions including: acute and chronic inflammation (Hofmannet al., Cell 97:889-901 (1999)), the development of diabetic latecomplications such as increased vascular permeability (Wautier et al.,J. Clin. Invest., 97:238-243 (1995)), nephropathy (Teillet et al., J.Am. Soc. Nephrol., 11:1488-1497 (2000)), arteriosclerosis (Vlassara et.al., The Finnish Medical Society DUODECIM, Ann. Med., 28:419-426(1996)), and retinopathy (Hammes et al., Diabetologia, 42:603-607(1999)). RAGE has also been implicated in Alzheimer's disease (Yan etal., Nature, 382:685-691 (1996)), and in tumor invasion and metastasis(Taguchi et al., Nature, 405:354-357 (2000)).

Despite the broad expression of RAGE and its apparent pleiotropic rolein multiple diverse disease models, RAGE does not appear to be essentialto normal development. For example, RAGE knockout mice are without anovert abnormal phenotype, suggesting that while RAGE can play a role indisease pathology when stimulated chronically, inhibition of RAGE doesnot appear to contribute to any unwanted acute phenotype (Liliensiek etal., J. Clin. Invest., 113:1641-50 (2004)).

Antagonizing binding of physiological ligands to RAGE may down-regulatethe pathophysiological changes brought about by excessive concentrationsof AGEs and other RAGE ligands. By reducing binding of endogenousligands to RAGE, symptoms associated with RAGE-mediated disorders may bereduced. Soluble RAGE (sRAGE) is able to effectively antagonize thebinding of RAGE ligands to RAGE. However, sRAGE can have a half-lifewhen administered in vivo that may be too short to be therapeuticallyuseful for one or more disorders. Thus, there is a need to developcompounds that antagonize the binding of AGEs and other physiologicalligands to the RAGE receptor where the compound has a desireablepharmacokinetic profile.

SUMMARY

Embodiments of the present invention comprise RAGE fusion proteins andmethods of using such proteins. The present invention may be embodied ina variety of ways. Embodiments of the present invention may comprise afusion protein comprising a RAGE polypeptide linked to a second,non-RAGE polypeptide. In one embodiment, the fusion protein comprises aRAGE ligand binding site. The fusion protein may further comprise a RAGEpolypeptide directly linked to a polypeptide comprising C_(H)2 domain ofan immunoglobulin, or a portion of the C_(H)2 domain.

The present invention also comprises a method to make a RAGE fusionprotein. In one embodiment the method comprises linking a RAGEpolypeptide to a second, non-RAGE polypeptide. In one embodiment, theRAGE polypeptide comprises a RAGE ligand binding site. The method maycomprise linking a RAGE polypeptide directly to a polypeptide comprisingthe C_(H)2 domain of an immunoglobulin or a portion of the C_(H)2domain.

In other embodiments, the present invention may comprise methods andcompositions for treating a RAGE-mediated disorder in a subject. Themethod may comprise administering a fusion protein of the presentinvention to the subject. The composition may comprise a RAGE fusionprotein of the present invention in a pharmaceutically acceptablecarrier.

There are various advantages that may be associated with particularembodiments of the present invention. In one embodiment, the fusionproteins of the present invention may be metabolically stable whenadministered to a subject. Also, the fusion proteins of the presentinvention may exhibit high-affinity binding for RAGE ligands. In certainembodiments, the fusion proteins of the present invention bind to RAGEligands with affinities in the high nanomolar to low micromolar range.By binding with high affinity to physiological RAGE ligands, the fusionproteins of the present invention may be used to inhibit binding ofendogenous ligands to RAGE, thereby providing a means to ameliorateRAGE-mediated diseases.

Also, the fusion proteins of the present invention may be provided inprotein or nucleic acid form. In one example embodiment, the fusionprotein may be administered systemically and remain in the vasculatureto potentially treat vascular diseases mediated in part by RAGE. Inanother example embodiment, the fusion protein may be administeredlocally to treat diseases where RAGE ligands contribute to the pathologyof the disease. Alternatively, a nucleic acid construct encoding thefusion protein may be delivered to a site by the use of an appropriatecarrier such as a virus or naked DNA where transient local expressionmay locally inhibit the interaction between RAGE ligands and receptors.Thus, administration may be transient (e.g., as where the fusion proteinis administered) or more permanent in nature (e.g., as where the fusionprotein is administered as a recombinant DNA).

There are additional features of the invention which will be describedhereinafter. It is to be understood that the invention is not limited inits application to the details set forth in the following claims,description and figures. The invention is capable of other embodimentsand of being practiced or carried out in various ways.

BRIEF DESCRIPTION OF THE FIGURES

Various features, aspects and advantages of the present invention willbecome more apparent with reference to the following figures.

FIG. 1 shows various RAGE sequences in accordance with alternateembodiments of the present invention: Panel A, SEQ ID NO: 1, the aminoacid sequence for human RAGE; and SEQ ID NO: 2, the amino acid sequencefor human RAGE without the signal sequence of amino acids 1-22; Panel B,SEQ ID NO: 3, the amino acid sequence for human RAGE without the signalsequence of amino acids 1-23; Panel C, SEQ ID NO: 4, the amino acidsequence of human sRAGE; SEQ ID NO: 5, the amino acid sequence of humansRAGE without the signal sequence of amino acids 1-22, and SEQ ID NO: 6,the amino acid sequence of human sRAGE without the signal sequence ofamino acids 1-23; Panel D, SEQ ID NO: 7, an amino acid sequencecomprising the V-domain of human RAGE; SEQ ID NO: 8, an alternate aminoacid sequence comprising the V-domain of human RAGE; SEQ ID NO: 9, anN-terminal fragment of the V-domain of human RAGE; SEQ ID NO: 10, analternate N-terminal fragment of the V-domain of human RAGE; SEQ ID NO:11, the amino acid sequence for amino acids 124-221 of human RAGE; SEQID NO: 12, the amino acid sequence for amino acids 227-317 of humanRAGE; SEQ ID NO: 13, the amino acid sequence for amino acids 23-123 ofhuman RAGE; Panel E, SEQ ID NO: 14, the amino acid sequence for aminoacids 24-123 of human RAGE; SEQ ID NO: 15, the amino acid sequence foramino acids 23-136 of human RAGE; SEQ ID NO: 16, the amino acid sequencefor amino acids 24-136 of human RAGE; SEQ ID NO: 17, the amino acidsequence for amino acids 23-226 of human RAGE; SEQ ID NO: 18, the aminoacid sequence for amino acids 24-226 of human RAGE; Panel F, SEQ ID NO:19, the amino acid sequence for amino acids 23-251 of human RAGE; SEQ IDNO: 20, the amino acid sequence for amino acids 24-251 of human RAGE;SEQ ID NO: 21, a RAGE interdomain linker; SEQ ID NO: 22, a second RAGEinterdomain linker; SEQ ID NO: 23, a third RAGE interdomain linker; SEQID NO: 24, a fourth RAGE interdomain linker; Panel G, SEQ ID NO: 25, DNAencoding human RAGE amino acids 1-118; SEQ ID NO: 26, DNA encoding humanRAGE amino acids 1-123; and SEQ ID NO: 27, DNA encoding human RAGE aminoacids 1-136; Panel H, SEQ ID NO: 28, DNA encoding human RAGE amino acids1-230; and SEQ ID NO: 29, DNA encoding human RAGE amino acids 1-251;Panel I, SEQ ID NO: 38, a partial amino acid sequence for the C_(H)2 andC_(H)3 domains of human IgG; SEQ ID NO:39, DNA encoding a portion of thehuman C_(H)2 and C_(H)3 domains of human IgG; SEQ ID NO: 40, an aminoacid sequence for the C_(H)2 and C_(H)3 domains of human IgG; Panel J,SEQ ID NO: 41, a DNA encoding the human C_(H)2 and C_(H)3 domains ofhuman IgG; SEQ ID NO: 42, an amino acid sequence for the C_(H)2 domainof human IgG; SEQ ID NO: 43, an amino acid sequence for the C_(H)3domain of human IgG; and SEQ ID NO: 44, a fifth RAGE interdomain linker.

FIG. 2 shows the DNA sequence (SEQ ID NO: 30) of a RAGE fusion protein(TTP-4000) coding region in accordance with an embodiment of the presentinvention. Coding sequence 1-753 highlighted in bold encodes RAGEN-terminal protein sequence whereas sequence 754-1386 encodes human IgGFc (γ1) protein sequence.

FIG. 3 shows the DNA sequence (SEQ ID NO: 31) of an alternate RAGEfusion protein (TTP-3000) coding region in accordance with an embodimentof the present invention. Coding sequence 1-408 highlighted in boldencodes RAGE N-terminal protein sequence, whereas sequence 409-1041codes human IgG Fc (γ1) protein sequence.

FIG. 4 shows the amino acid sequences, SEQ ID NO: 32 (TTP-4000), SEQ IDNO: 33, and SEQ ID NO: 34, that each encode a four domain RAGE fusionprotein in accordance with alternate embodiments of the presentinvention. RAGE sequence is highlighted with bold font.

FIG. 5 shows the amino acid sequences, SEQ ID NO: 35 (TTP-3000), SEQ IDNO: 36, and SEQ ID NO: 37, that each encode a three domain RAGE fusionprotein in accordance with alternate embodiments of the presentinvention. RAGE sequence is highlighted with bold font.

FIG. 6, Panel A, shows a comparison of the protein domains in human RAGEand human Ig gamma-1 Fc protein, and cleavage points used to makeTTP-3000 (at position 136) and TTP-4000 (at position 251) in accordancewith alternate embodiments of the present invention; and Panel B showsthe domain structure for TTP-3000 and TTP-4000 in accordance withalternate embodiments of the present invention.

FIG. 7 shows results of an in vitro binding assay for sRAGE, and RAGEfusion proteins TTP-4000 (TT4) and TTP-3000 (TT3), to the RAGE ligandsamyloid-beta (A-beta), S100b (S100), and amphoterin (Ampho), inaccordance with an embodiment of the present invention.

FIG. 8 shows results of an in vitro binding assay for RAGE fusionprotein TTP-4000 (TT4) (“Protein”) to amyloid-beta as compared to anegative control only including the immunodetection reagents (“ComplexAlone”), and antagonism of such binding by a RAGE antagonist (“RAGELigand”) in accordance with an embodiment of the present invention.

FIG. 9 shows results of an in vitro binding assay for RAGE fusionprotein TTP-3000 (TT3) (“Protein”) to amyloid-beta as compared to anegative control only including the immunodetection reagents (“ComplexAlone”), and antagonism of such binding by a RAGE antagonist (“RAGELigand”) in accordance with an embodiment of the present invention.

FIG. 10 shows results of a cell-based assay measuring the inhibition ofS100b-RAGE induced production of TNF-α by RAGE fusion proteins TTP-3000(TT3) and TTP-4000 (TT4), and sRAGE in accordance with an embodiment ofthe present invention.

FIG. 11 shows a pharmacokinetic profile for RAGE fusion protein TTP-4000in accordance with an embodiment of the present invention wherein eachcurve represents a different animal under the same experimentalconditions.

FIG. 12 shows relative levels of TNF-α release from THP-1 cells due tostimulation by RAGE fusion protein TTP-4000 and human IgG stimulation asa measure of an inflammatory response in accordance with an embodimentof the present invention FIG. 13 shows the use of RAGE fusion proteinTTP-4000 to reduce restenosis in diabetic animals in accordance withalternate embodiments of the present invention, wherein panel A showsthat TTP-4000 RAGE-fusion protein reduced the intima/media ratio ascompared to a negative control (IgG), and panel B shows that TTP-4000RAGE-fusion protein reduced vascular smooth muscle cell proliferation ina dose-responsive manner.

FIG. 14 shows use of RAGE fusion protein TTP-4000 to reduce amyloidformation and cognitive dysfunction in animals with Alzheimer's Disease(AD) in accordance with alternate embodiments of the present inventionwherein panel A shows TTP-4000 RAGE-fusion protein reduced amyloid loadin the brain, and panel B shows TTP-4000 RAGE-fusion protein improvedcognitive function.

FIG. 15 shows saturation-binding curves with TTP-4000 to variousimmobilized known RAGE ligands in accordance with an embodiment of thepresent invention.

DETAILED DESCRIPTION

For the purposes of this specification, unless otherwise indicated, allnumbers expressing quantities of ingredients, reaction conditions, andso forth used in the specification are to be understood as beingmodified in all instances by the term “about.” Accordingly, unlessindicated to the contrary, the numerical parameters set forth in thefollowing specification are approximations that can vary depending uponthe desired properties sought to be obtained by the present invention.At the very least, and not as an attempt to limit the application of thedoctrine of equivalents to the scope of the claims, each numericalparameter should at least be construed in light of the number ofreported significant digits and by applying ordinary roundingtechniques.

Notwithstanding that the numerical ranges and parameters setting forththe broad scope of the invention are approximations, the numericalvalues set forth in the specific examples are reported as precisely aspossible. Any numerical value, however, inherently contains certainerrors necessarily resulting from the standard deviation found in theirrespective testing measurements. Moreover, all ranges disclosed hereinare to be understood to encompass any and all subranges subsumedtherein. For example, a stated range of “1 to 10” should be consideredto include any and all subranges between (and inclusive of) the minimumvalue of 1 and the maximum value of 10; that is, all subranges beginningwith a minimum value of 1 or more, e.g. 1 to 6.1, and ending with amaximum value of 10 or less, e.g., 5.5 to 10. Additionally, anyreference referred to as being “incorporated herein” is to be understoodas being incorporated in its entirety.

It is further noted that, as used in this specification, the singularforms “a,” “an,” and “the” include plural referents unless expressly andunequivocally limited to one referent. The term “or” is usedinterchangeably with the term “and/or” unless the context clearlyindicates otherwise.

Also, the terms “portion” and “fragment” are used interchangeably torefer to parts of a polypeptide, nucleic acid, or other molecularconstruct.

As used herein, the term “upstream” refers to a residue that isN-terminal to a second residue where the molecule is a protein, or 5′ toa second residue where the molecule is a nucleic acid. Also as usedherein, the term “downstream” refers to a residue that is C-terminal toa second residue where the molecule is a protein, or 3′ to a secondresidue where the molecule is a nucleic acid.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art. Practitioners are particularly directed to Current Protocols inMolecular Biology (Ansubel) for definitions and terms of the art.Abbreviations for amino acid residues are the standard 3-letter and/or1-letter codes used in the art to refer to one of the 20 common L-aminoacids.

A “nucleic acid” is a polynucleotide such as deoxyribonucleic acid (DNA)or ribonucleic acid (RNA). The term is used to include single-strandednucleic acids, double-stranded nucleic acids, and RNA and DNA made fromnucleotide or nucleoside analogues.

The term “vector” refers to a nucleic acid molecule that may be used totransport a second nucleic acid molecule into a cell. In one embodiment,the vector allows for replication of DNA sequences inserted into thevector. The vector may comprise a promoter to enhance expression of thenucleic acid molecule in at least some host cells. Vectors may replicateautonomously (extrachromasomal) or may be integrated into a host cellchromosome. In one embodiment, the vector may comprise an expressionvector capable of producing a protein derived from at least part of anucleic acid sequence inserted into the vector.

As is known in the art, conditions for hybridizing nucleic acidsequences to each other can be described as ranging from low to highstringency. Generally, highly stringent hybridization conditions referto washing hybrids in low salt buffer at high temperatures.Hybridization may be to filter bound DNA using hybridization solutionsstandard in the art such as 0.5M NaHPO₄, 7% sodium dodecyl sulfate(SDS), at 65° C., and washing in 0.25 M NaHPO₄, 3.5% SDS followed bywashing 0.1×SSC/0.1% SDS at a temperature ranging from room temperatureto 68° C. depending on the length of the probe (see e.g. Ausubel, F. M.et al., Short Protocols in Molecular Biology, 4^(th) Ed., Chapter 2,John Wiley & Sons, N.Y). For example, a high stringency wash compriseswashing in 6×SSC/0.05% sodium pyrophosphate at 37° C. for a 14 baseoligonucleotide probe, or at 48° C. for a 17 base oligonucleotide probe,or at 55° C. for a 20 base oligonucleotide probe, or at 60° C. for a 25base oligonucleotide probe, or at 65° C. for a nucleotide probe about250 nucleotides in length. Nucleic acid probes may be labeled withradionucleotides by end-labeling with, for example, [γ-³²P]ATP, orincorporation of radiolabeled nucleotides such as [α-³²P]dCTP by randomprimer labeling. Alternatively, probes may be labeled by incorporationof biotinylated or fluorescein labeled nucleotides, and the probedetected using Streptavidin or anti-fluorescein antibodies.

As used herein, “small organic molecules” are molecules of molecularweight less than 2,000 Daltons that contain at least one carbon atom.

“Polypeptide” and “protein” are used interchangeably herein to describeprotein molecules that may comprise either partial or full-lengthproteins.

The term “fusion protein” refers to a protein or polypeptide that has anamino acid sequence derived from two or more proteins. The fusionprotein may also include linking regions of amino acids between aminoacid portions derived from separate proteins.

As used herein, a “non-RAGE polypeptide” is any polypeptide that is notderived from RAGE or a fragment thereof. Such non-RAGE polypeptidesinclude immunoglobulin peptides, dimerizing polypeptides, stabilizingpolypeptides, amphiphilic peptides, or polypeptides comprising aminoacid sequences that provide “tags” for targeting or purification of theprotein.

As used herein, “immunoglobulin peptides” may comprise an immunoglobulinheavy chain or a portion thereof. In one embodiment, the portion of theheavy chain may be the Fc fragment or a portion thereof. As used herein,the Fc fragment comprises the heavy chain hinge polypeptide, and theC_(H)2 and C_(H)3 domains of the heavy chain of an immunoglobulin, ineither monomeric or dimeric form. Or, the C_(H)1 and Fc fragment may beused as the immunoglobulin polypeptide. The heavy chain (or portionthereof) may be derived from any one of the known heavy chain isotypes:IgG (γ), IgM (μ), IgD (δ), IgE (ε), or IgA (α). In addition, the heavychain (or portion thereof) may be derived from any one of the knownheavy chain subtypes: IgG1 (γ1), IgG2 (γ2), IgG3 (γ3), IgG4 (γ4), IgA1(α1), IgA2 (α2), or mutations of these isotypes or subtypes that alterthe biological activity. An example of biological activity that may bealtered includes reduction of an isotype's ability to bind to some Fcreceptors as for example, by modification of the hinge region.

The terms “identity” or “percent identical” refers to sequence identitybetween two amino acid sequences or between two nucleic acid sequences.Percent identity can be determined by aligning two sequences and refersto the number of identical residues (i.e., amino acid or nucleotide) atpositions shared by the compared sequences. Sequence alignment andcomparison may be conducted using the algorithms standard in the art(e.g. Smith and Waterman, 1981, Adv. Appl. Math. 2:482; Needleman andWunsch, 1970, J. Mol. Biol. 48:443; Pearson and Lipman, 1988, Proc.Natl. Acad. Sci., USA, 85:2444) or by computerized versions of thesealgorithms (Wisconsin Genetics Software Package Release 7.0, GeneticsComputer Group, 575 Science Drive, Madison, Wis.) publicly available asBLAST and FASTA. Also, ENTREZ, available through the National Institutesof Health, Bethesda Md., may be used for sequence comparison. In oneembodiment, the percent identity of two sequences may be determinedusing GCG with a gap weight of 1, such that each amino acid gap isweighted as if it were a single amino acid mismatch between the twosequences.

As used herein, the term “conserved residues” refers to amino acids thatare the same among a plurality of proteins having the same structureand/or function. A region of conserved residues may be important forprotein structure or function. Thus, contiguous conserved residues asidentified in a three-dimensional protein may be important for proteinstructure or function. To find conserved residues, or conserved regionsof 3-D structure, a comparison of sequences for the same or similarproteins from different species, or of individuals of the same species,may be made.

As used herein, the term “homologue” means a polypeptide having a degreeof homology with the wild-type amino acid sequence. Homology comparisonscan be conducted by eye, or more usually, with the aid of readilyavailable sequence comparison programs. These commercially availablecomputer programs can calculate percent homology between two or moresequences (e.g. Wilbur, W. J. and Lipman, D. J., 1983, Proc. Natl. Acad.Sci. USA, 80:726-730). For example, homologous sequences may be taken toinclude an amino acid sequences which in alternate embodiments are atleast 75% identical, 85% identical, 90% identical, 95% identical, or 98%identical to each other.

As used herein, a polypeptide or protein “domain” comprises a regionalong a polypeptide or protein that comprises an independent unit.Domains may be defined in terms of structure, sequence and/or biologicalactivity. In one embodiment, a polypeptide domain may comprise a regionof a protein that folds in a manner that is substantially independentfrom the rest of the protein. Domains may be identified using domaindatabases such as, but not limited to PFAM, PRODOM, PROSITE, BLOCKS,PRINTS, SBASE, ISREC PROFILES, SAMRT, and PROCLASS.

As used herein, “immunoglobulin domain” is a sequence of amino acidsthat is structurally homologous, or identical to, a domain of animmunoglobulin. The length of the sequence of amino acids of animmunoglobulin domain may be any length. In one embodiment, animmunoglobulin domain may be less than 250 amino acids. In an exampleembodiment, an immunoglobulin domain may be about 80-150 amino acids inlength. For example, the variable region, and the C_(H)1, C_(H)2, andC_(H)3 regions of an IgG are each immunoglobulin domains. In anotherexample, the variable, the C_(H)1, C_(H)2, C_(H)3 and C_(H)4 regions ofan IgM are each immunoglobulin domains.

As used herein, a “RAGE immunoglobulin domain” is a sequence of aminoacids from RAGE protein that is structurally homologous, or identicalto, a domain of an immunoglobulin. For example, a RAGE immunoglobulindomain may comprise the RAGE V-domain, the RAGE Ig-like C2-type 1 domain(“C1 domain”), or the RAGE Ig-like C2-type 2 domain (“C2 domain”).

As used herein, an “interdomain linker” comprises a polypeptide thatjoins two domains together. An Fc hinge region is an example of aninterdomain linker in an IgG.

As used herein, “directly linked” identifies a covalent linkage betweentwo different groups (e.g., nucleic acid sequences, polypeptides,polypeptide domains) that does not have any intervening atoms betweenthe two groups that are being linked.

As used herein, “ligand binding domain” refers to a domain of a proteinresponsible for binding a ligand. The term ligand binding domainincludes homologues of a ligand binding domain or portions thereof. Inthis regard, deliberate amino acid substitutions may be made in theligand binding site on the basis of similarity in polarity, charge,solubility, hydrophobicity, or hydrophilicity of the residues, as longas the binding specificity of the ligand binding domain is retained.

As used herein, a “ligand binding site” comprises residues in a proteinthat directly interact with a ligand, or residues involved inpositioning the ligand in close proximity to those residues thatdirectly interact with the ligand. The interaction of residues in theligand binding site may be defined by the spatial proximity of theresidues to a ligand in the model or structure. The term ligand bindingsite includes homologues of a ligand binding site, or portions thereof.In this regard, deliberate amino acid substitutions may be made in theligand binding site on the basis of similarity in polarity, charge,solubility, hydrophobicity, or hydrophilicity of the residues, as longas the binding specificity of the ligand binding site is retained. Aligand binding site may exist in one or more ligand binding domains of aprotein or polypeptide.

As used herein, the term “interact” refers to a condition of proximitybetween a ligand or compound, or portions or fragments thereof, and aportion of a second molecule of interest. The interaction may benon-covalent, for example, as a result of hydrogen-bonding, van derWaals interactions, or electrostatic or hydrophobic interactions, or itmay be covalent.

As used herein, a “ligand” refers to a molecule or compound or entitythat interacts with a ligand binding site, including substrates oranalogues or parts thereof. As described herein, the term “ligand” mayrefer to compounds that bind to the protein of interest. A ligand may bean agonist, an antagonist, or a modulator. Or, a ligand may not have abiological effect. Or, a ligand may block the binding of other ligandsthereby inhibiting a biological effect. Ligands may include, but are notlimited to, small molecule inhibitors. These small molecules may includepeptides, peptidomimetics, organic compounds and the like. Ligands mayalso include polypeptides and/or proteins.

As used herein, a “modulator compound” refers to a molecule whichchanges or alters the biological activity of a molecule of interest. Amodulator compound may increase or decrease activity, or change thephysical or chemical characteristics, or functional or immunologicalproperties, of the molecule of interest. For RAGE, a modulator compoundmay increase or decrease activity, or change the characteristics, orfunctional or immunological properties of the RAGE, or a portion threofA modulator compound may include natural and/or chemically synthesizedor artificial peptides, modified peptides (e.g., phosphopeptides),antibodies, carbohydrates, monosaccharides, oligosaccharides,polysaccharides, glycolipids, heterocyclic compounds, nucleosides ornucleotides or parts thereof, and small organic or inorganic molecules.A modulator compound may be an endogenous physiological compound or itmay be a natural or synthetic compound. Or, the modulator compound maybe a small organic molecule. The term “modulator compound” also includesa chemically modified ligand or compound, and includes isomers andracemic forms.

An “agonist” comprises a compound that binds to a receptor to form acomplex that elicits a pharmacological response specific to the receptorinvolved.

An “antagonist” comprises a compound that binds to an agonist or to areceptor to form a complex that does not give rise to a substantialpharmacological response and can inhibit the biological response inducedby an agonist.

RAGE agonists may therefore bind to RAGE and stimulate RAGE-mediatedcellular processes, and RAGE antagonists may inhibit RAGE-mediatedprocesses from being stimulated by a RAGE agonist. For example, in oneembodiment, the cellular process stimulated by RAGE agonists comprisesactivation of TNF-α gene transcription.

The term “peptide mimetics” refers to structures that serve assubstitutes for peptides in interactions between molecules (Morgan etal., 1989, Ann. Reports Med. Chem., 24:243-252). Peptide mimetics mayinclude synthetic structures that may or may not contain amino acidsand/or peptide bonds but that retain the structural and functionalfeatures of a peptide, or agonist, or antagonist. Peptide mimetics alsoinclude peptoids, oligopeptoids (Simon et al., 1972, Proc. Natl. Acad,Sci., USA, 89:9367); and peptide libraries containing peptides of adesigned length representing all possible sequences of amino acidscorresponding to a peptide, or agonist or antagonist of the invention.

The term “treating” refers to improving a symptom of a disease ordisorder and may comprise curing the disorder, substantially preventingthe onset of the disorder, or improving the subject's condition. Theterm “treatment” as used herein, refers to the full spectrum oftreatments for a given disorder from which the patient is suffering,including alleviation of one symptom or most of the symptoms resultingfrom that disorder, a cure for the particular disorder, or prevention ofthe onset of the disorder.

As used herein, the term “EC50” is defined as the concentration of anagent that results in 50% of a measured biological effect. For example,the EC50 of a therapeutic agent having a measurable biological effectmay comprise the value at which the agent displays 50% of the biologicaleffect.

As used herein, the term “IC50” is defined as the concentration of anagent that results in 50% inhibition of a measured effect. For example,the IC50 of an antagonist of RAGE binding may comprise the value atwhich the antagonist reduces ligand binding to the ligand binding siteof RAGE by 50%.

As used herein, an “effective amount” means the amount of an agent thatis effective for producing a desired effect in a subject. The term“therapeutically effective amount” denotes that amount of a drug orpharmaceutical agent that will elicit therapeutic response of an animalor human that is being sought. The actual dose which comprises theeffective amount may depend upon the route of administration, the sizeand health of the subject, the disorder being treated, and the like.

The term “pharmaceutically acceptable carrier” as used herein may referto compounds and compositions that are suitable for use in human oranimal subjects, as for example, for therapeutic compositionsadministered for the treatment of a RAGE-mediated disorder or disease.

The term “pharmaceutical composition” is used herein to denote acomposition that may be administered to a mammalian host, e.g., orally,parenterally, topically, by inhalation spray, intranasally, or rectally,in unit dosage formulations containing conventional non-toxic carriers,diluents, adjuvants, vehicles and the like.

The term “parenteral” as used herein, includes subcutaneous injections,intravenous, intramuscular, intracisternal injection, or infusiontechniques.

RAGE Fusion Proteins

Embodiments of the present invention comprise RAGE fusion proteins,methods of making such fusion proteins, and methods of use of suchfusion proteins. The present invention may be embodied in a variety ofways.

For example, embodiments of the present invention provide fusionproteins comprising a RAGE polypeptide linked to a second, non-RAGEpolypeptide. In one embodiment, the fusion protein may comprise a RAGEligand binding site. In an embodiment, the ligand binding site comprisesthe most N-terminal domain of the fusion protein. The RAGE ligandbinding site may comprise the V domain of RAGE, or a portion thereof. Inan embodiment, the RAGE ligand binding site comprises SEQ ID NO: 9 or asequence 90% identical thereto, or SEQ ID NO: 10 or a sequence 90%identical thereto.

In an embodiment, the RAGE polypeptide may be linked to a polypeptidecomprising an immunoglobulin domain or a portion (e.g., a fragmentthereof) of an immunoglobulin domain. In one embodiment, the polypeptidecomprising an immunoglobulin domain comprises at least a portion of atleast one of the C_(H)2 or the C_(H)3 domains of a human IgG.

A RAGE protein or polypeptide may comprise full-length human RAGEprotein (e.g., SEQ ID NO: 1), or a fragment of human RAGE. As usedherein, a fragment of a RAGE polypeptide is at least 5 amino acids inlength, may be greater than 30 amino acids in length, but is less thanthe full amino acid sequence. In alternate embodiments, the RAGEpolypeptide may comprise a sequence that is 70%, or 80%, or 85%, or 90%identical to human RAGE, or a fragment thereof. For example, in oneembodiment, the RAGE polypeptide may comprise human RAGE, or a fragmentthereof, with Glycine as the first residue rather than a Methionine (seee.g., Neeper et al., (1992)). Or, the human RAGE may comprisefull-length RAGE with the signal sequence removed (e.g., SEQ ID NO: 2 orSEQ ID NO: 3) (FIGS. 1A and 1B) or a portion of that amino acidsequence.

The fusion proteins of the present invention may also comprise sRAGE(e.g., SEQ ID NO: 4), a polypeptide 90% identical to sRAGE, or afragment of sRAGE. As used herein, sRAGE is the RAGE protein that doesnot include the transmembrane region or the cytoplasmic tail (Park etal., Nature Med., 4:1025-1031 (1998)). For example, the RAGE polypeptidemay comprise human sRAGE, or a fragment thereof, with Glycine as thefirst residue rather than a Methionine (see e.g., Neeper et al.,(1992)). Or, a RAGE polypeptide may comprise human sRAGE with the signalsequence removed (e.g., SEQ ID NO: 5 or SEQ ID NO: 6) (FIG. 1C) or aportion of that amino acid sequence.

In other embodiments, the RAGE protein may comprise a RAGE V domain(e.g., SEQ ID NO: 7 or SEQ ID NO: 8; FIG. 1D) (Neeper et al., (1992);Schmidt et al. (1997)). Or, a sequence 90% identical to the RAGE Vdomain or a fragment thereof may be used.

Or, the RAGE protein may comprise a fragment of the RAGE V domain (e.g.,SEQ ID NO: 9 or SEQ ID NO: 10, FIG. 1D). In one embodiment the RAGEprotein may comprise a ligand binding site. In an embodiment, the ligandbinding site may comprise SEQ ID NO: 9, or a sequence 90% identicalthereto, or SEQ ID NO: 10, or a sequence 90% identical thereto. In yetanother embodiment, the RAGE fragment is a synthetic peptide.

Thus, the RAGE polypeptide used in the fusion proteins of the presentinvention may comprise a fragment of full length RAGE. As is known inthe art, RAGE comprises three immunoglobulin-like polypeptide domains,the V domain, and the C1 and C2 domains each linked to each other by aninterdomain linker. Full-length RAGE also includes a transmembranepolypeptide and a cytoplasmic tail downstream (C-terminal) of the C2domain, and linked to the C2 domain.

In an embodiment, the RAGE polypeptide does not include any signalsequence residues. The signal sequence of RAGE may comprise eitherresidues 1-22 or residues 1-23 of full length RAGE.

For example, the RAGE polypeptide may comprise amino acids 23-116 ofhuman RAGE (SEQ ID NO: 7) or a sequence 90% identical thereto, or aminoacids 24-116 of human RAGE (SEQ ID NO: 8) or a sequence 90% identicalthereto, corresponding to the V domain of RAGE. Or, the RAGE polypeptidemay comprise amino acids 124-221 of human RAGE (SEQ ID NO: 11) or asequence 90% identical thereto, corresponding to the C1 domain of RAGE.In another embodiment, the RAGE polypeptide may comprise amino acids227-317 of human RAGE (SEQ ID NO: 12) or a sequence 90% identicalthereto, corresponding to the C2 domain of RAGE. Or, the RAGEpolypeptide may comprise amino acids 23-123 of human RAGE (SEQ ID NO:13) or a sequence 90% identical thereto, or amino acids 24-123 of humanRAGE (SEQ ID NO: 14) or a sequence 90% identical thereto, correspondingto the V domain of RAGE and a downstream interdomain linker. Or, theRAGE polypeptide may comprise amino acids 23-226 of human RAGE (SEQ IDNO: 17) or a sequence 90% identical thereto, or amino acids 24-226 ofhuman RAGE (SEQ ID NO: 18) or a sequence 90% identical thereto,corresponding to the V-domain, the C1 domain and the interdomain linkerlinking these two domains. Or, the RAGE polypeptide may comprise aminoacids 23-339 of human RAGE (SEQ ID NO: 5) or a sequence 90% identicalthereto, or 24-339 of human RAGE (SEQ ID NO: 6) or a sequence 90%identical thereto, corresponding to sRAGE (i.e., encoding the V, C1, andC2 domains and interdomain linkers). Or, fragments of each of thesesequences may be used.

The fusion protein may include several types of peptides that are notderived from RAGE or a fragment thereof. The second polypeptide of thefusion protein may comprise a polypeptide derived from animmunoglobulin. In one embodiment, the immunoglobulin polypeptide maycomprise an immunoglobulin heavy chain or a portion (i.e., fragment)thereof. For example, the heavy chain fragment may comprise apolypeptide derived from the Fc fragment of an immunoglobulin, whereinthe Fc fragment comprises the heavy chain hinge polypeptide, and C_(H)2and C_(H)3 domains of the immunoglobulin heavy chain as a monomer. Theheavy chain (or portion thereof) may be derived from any one of theknown heavy chain isotypes: IgG (γ), IgM (μ), IgD (δ), IgE (ε), or IgA(α). In addition, the heavy chain (or portion thereof) may be derivedfrom any one of the known heavy chain subtypes: IgG1 (γ1), IgG2 (γ2),IgG3 (γ3), IgG4 (γ4), IgA1 (α1), IgA2 (α2), or mutations of theseisotypes or subtypes that alter the biological activity. The secondpolypeptide may comprise the C_(H)2 and C_(H)3 domains of a human IgG1or portions of either, or both, of these domains. As an exampleembodiments, the polypeptide comprising the C_(H)2 and C_(H)3 domains ofa human IgG1 or a portion thereof may comprise SEQ ID NO: 38 or SEQ IDNO: 40.

The Fc portion of the immunoglobulin chain may be proinflammatory invivo. Thus, in one embodiment, the RAGE fusion protein of the presentinvention comprises an interdomain linker derived from RAGE rather thanan interdomain hinge polypeptide derived from an immunoglobulin.

Thus in one embodiment, the fusion protein may further comprise a RAGEpolypeptide directly linked to a polypeptide comprising a C_(H)2 domainof an immunoglobulin, or a fragment or portion of the C_(H)2 domain ofan immunoglobulin. In one embodiment, the C_(H)2 domain, or a fragmentthereof comprises SEQ ID NO: 42. In one embodiment, the RAGE polypeptidemay comprise a ligand binding site. The RAGE ligand binding site maycomprise the V domain of RAGE, or a portion thereof. In an embodiment,the RAGE ligand binding site comprises SEQ ID NO: 9 or a sequence 90%identical thereto, or SEQ ID NO: 10 or a sequence 90% identical thereto.

The RAGE polypeptide used in the fusion proteins of the presentinvention may comprise a RAGE immunoglobulin domain. Additionally oralternatively, the fragment of RAGE may comprise an interdomain linker.Or, the RAGE polypeptide may comprise a RAGE immunoglobulin domainlinked to an upstream (i.e., closer to the N-terminus) or downstream(i.e., closer to the C-terminus) interdomain linker. In yet anotherembodiment, the RAGE polypeptide may comprise two (or more) RAGEimmunoglobulin domains each linked to each other by an interdomainlinker. The RAGE polypeptide may further comprise multiple RAGEimmunoglobulin domains linked to each other by one or more interdomainlinkers and having a terminal interdomain linker attached to theN-terminal RAGE immunoglobulin domain and/or the C-terminalimmunoglobulin domain. Additional combinations of RAGE immunoglobulindomains and interdomain linkers are within the scope of the presentinvention.

In one embodiment, the RAGE polypeptide comprises a RAGE interdomainlinker linked to a RAGE immunoglobulin domain such that the C-terminalamino acid of the RAGE immunoglobulin domain is linked to the N-terminalamino acid of the interdomain linker, and the C-terminal amino acid ofthe RAGE interdomain linker is directly linked to the N-terminal aminoacid of a polypeptide comprising a C_(H)2 domain of an immunoglobulin,or a fragment thereof. The polypeptide comprising a C_(H)2 domain of animmunoglobulin may comprise the C_(H)2 and C_(H)3 domains of a humanIgG1 or a portion of either, or both, of these domains. As an exampleembodiment, the polypeptide comprising the C_(H)2 and C_(H)3 domains, ora portion thereof, of a human IgG1 may comprise SEQ ID NO: 38 or SEQ IDNO: 40.

As described above, the fusion protein of the present invention maycomprise a single or multiple domains from RAGE. Also, the RAGEpolypeptide comprising an interdomain linker linked to a RAGEpolypeptide domain may comprise a fragment of full-length RAGE protein.For example, the RAGE polypeptide may comprise amino acids 23-136 ofhuman RAGE (SEQ ID NO: 15) or a sequence 90% identical thereto or aminoacids 24-136 of human RAGE (SEQ ID NO: 16) or a sequence 90% identicalthereto corresponding to the V domain of RAGE and a downstreaminterdomain linker. Or, the RAGE polypeptide may comprise amino acids23-251 of human RAGE (SEQ ID NO: 19) or a sequence 90% identicalthereto, or amino acids 24-251 of human RAGE (SEQ ID NO: 20) or asequence 90% identical thereto, corresponding to the V-domain, the C1domain, the interdomain linker linking these two domains, and a secondinterdomain linker downstream of C1.

For example, in one embodiment, the fusion protein may comprise twoimmunoglobulin domains derived from RAGE protein and two immunoglobulindomains derived from a human Fc polypeptide. The fusion protein maycomprise a first RAGE immunoglobulin domain and a first RAGE interdomainlinker linked to a second RAGE immunoglobulin domain and a second RAGEinterdomain linker, such that the N-terminal amino acid of the firstinterdomain linker is linked to the C-terminal amino acid of the firstRAGE immunoglobulin domain, the N-terminal amino acid of the second RAGEimmunoglobulin domain is linked to C-terminal amino acid of the firstinterdomain linker, the N-terminal amino acid of the second interdomainlinker is linked to C-terminal amino acid of the second RAGEimmunoglobulin domain, and the C-terminal amino acid of the RAGE secondinterdomain linker is directly linked to the N-terminal amino acid ofthe C_(H)2 immunoglobulin domain. In one embodiment, a four domain RAGEfusion protein may comprise SEQ ID NO: 32. In alternate embodiments, afour domain RAGE fusion protein comprises SEQ ID NO: 33 or SEQ ID NO:34.

Alternatively, a three domain fusion protein may comprise oneimmunoglobulin domain derived from RAGE and two immunoglobulin domainsderived from a human Fc polypeptide. For example, the fusion protein maycomprise a single RAGE immunoglobulin domain linked via a RAGEinterdomain linker to the N-terminal amino acid of a C_(H)2immunoglobulin domain or a portion of a C_(H)2 immunoglobulin domain. Inone embodiment, a three domain RAGE fusion protein may comprise SEQ IDNO: 35. In alternate embodiments, a three domain RAGE fusion protein maycomprise SEQ ID NO: 36 or SEQ ID NO: 37.

A RAGE interdomain linker fragment may comprise a peptide sequence thatis naturally downstream of, and thus, linked to, a RAGE immunoglobulindomain. For example, for the RAGE V domain, the interdomain linker maycomprise amino acid sequences that are naturally downstream from the Vdomain. In an embodiment, the linker may comprise SEQ ID NO: 21,corresponding to amino acids 117-123 of full-length RAGE. Or, the linkermay comprise a peptide having additional portions of the natural RAGEsequence. For example, a interdomain linker comprising several aminoacids (e.g., 1-3, 1-5, or 1-10, or 1-15 amino acids) upstream anddownstream of SEQ ID NO: 21 may be used. Thus, in one embodiment, theinterdomain linker comprises SEQ ID NO: 23 comprising amino acids117-136 of full-length RAGE. Or, fragments of SEQ ID NO: 21 deleting,for example, 1, 2, or 3, amino acids from either end of the linker maybe used. In alternate embodiments, the linker may comprise a sequencethat is 70% identical, or 80% identical, or 90% identical to SEQ ID NO:21 or SEQ ID NO: 23.

For the RAGE C1 domain, the linker may comprise peptide sequence that isnaturally downstream of the C1 domain. In an embodiment, the linker maycomprise SEQ ID NO: 22, corresponding to amino acids 222-251 offull-length RAGE. Or, the linker may comprise a peptide havingadditional portions of the natural RAGE sequence. For example, a linkercomprising several (1-3, 1-5, or 1-10, or 1-15 amino acids) amino acidsupstream and downstream of SEQ ID NO: 22 may be used. Or, fragments ofSEQ ID NO: 22 may be used, deleting for example, 1-3, 1-5, or 1-10, or1-15 amino acids from either end of the linker. For example, in oneembodiment, a RAGE interdomain linker may comprise SEQ ID NO: 24,corresponding to amino acids 222-226. Or an interdomain linker maycomprise SEQ ID NO: 44, corresponding to RAGE amino acids 318-342.

Methods of Producing RAGE Fusion Proteins

The present invention also comprises a method to make a RAGE fusionprotein. Thus, in one embodiment, the present invention comprises amethod of making a RAGE fusion protein comprising the step of covalentlylinking a RAGE polypeptide linked to a second, non-RAGE polypeptidewherein the RAGE polypeptide comprises a RAGE ligand binding site. Forexample, the linked RAGE polypeptide and the second, non-RAGEpolypeptide may be encoded by a recombinant DNA construct. The methodmay further comprise the step of incorporating the DNA construct into anexpression vector. Also, the method may comprise the step of insertingthe expression vector into a host cell.

For example, embodiments of the present invention provide fusionproteins comprising a RAGE polypeptide linked to a second, non-RAGEpolypeptide. In one embodiment, the fusion protein may comprise a RAGEligand binding site. In an embodiment, the ligand binding site comprisesthe most N-terminal domain of the fusion protein. The RAGE ligandbinding site may comprise the V domain of RAGE, or a portion thereof. Inan embodiment, the RAGE ligand binding site comprises SEQ ID NO: 9 or asequence 90% identical thereto, or SEQ ID NO: 10 or a sequence 90%identical thereto.

In an embodiment, the RAGE polypeptide may be linked to a polypeptidecomprising an immunoglobulin domain or a portion (e.g., a fragmentthereof) of an immunoglobulin domain. In one embodiment, the polypeptidecomprising an immunoglobulin domain comprises at least a portion of atleast one of the C_(H)2 or the C_(H)3 domains of a human IgG.

The fusion protein may be engineered by recombinant DNA techniques. Forexample, in one embodiment, the present invention may comprise anisolated nucleic acid sequence encoding a RAGE polypeptide linked to asecond, non-RAGE polypeptide. In an embodiment, the RAGE polypeptide maycomprise a RAGE ligand binding site.

The RAGE protein or polypeptide may comprise full-length human RAGE(e.g., SEQ ID NO: 1), or a fragment of human RAGE. In an embodiment, theRAGE polypeptide does not include any signal sequence residues. Thesignal sequence of RAGE may comprise either residues 1-22 or residues1-23 of full length RAGE (SEQ ID NO: 1). In alternate embodiments, theRAGE polypeptide may comprise a sequence 70%, or 80%, or 90% identicalto human RAGE, or a fragment thereof. For example, in one embodiment,the RAGE polypeptide may comprise human RAGE, or a fragment thereof,with Glycine as the first residue rather than a Methionine (see e.g.,Neeper et al., (1992)). Or, the human RAGE may comprise full-length RAGEwith the signal sequence removed (e.g., SEQ ID NO: 2 or SEQ ID NO: 3)(FIGS. 1A and 1B) or a portion of that amino acid sequence. The fusionproteins of the present invention may also comprise sRAGE (e.g., SEQ IDNO: 4), a polypeptide 90% identical to sRAGE, or a fragment of sRAGE.For example, the RAGE polypeptide may comprise human sRAGE, or afragment thereof, with Glycine as the first residue rather than aMethionine (see e.g., Neeper et al., (1992)). Or, the human RAGE maycomprise sRAGE with the signal sequence removed (e.g., SEQ ID NO: 5 orSEQ ID NO: 6) (FIG. 1C) or a portion of that amino acid sequence. Inother embodiments, the RAGE protein may comprise a V domain (e.g., SEQID NO: 7 or SEQ ID NO: 8; FIG. 1D). Or, a sequence 90% identical to theV domain or a fragment thereof may be used. Or, the RAGE protein maycomprise a fragment of RAGE comprising a portion of the V domain (e.g.,SEQ ID NO: 9 or SEQ ID NO: 10, FIG. 1D). In an embodiment, the ligandbinding site may comprise SEQ ID NO: 9, or a sequence 90% identicalthereto, or SEQ ID NO: 10, or a sequence 90% identical thereto. In yetanother embodiment, the RAGE fragment is a synthetic peptide.

In an embodiment, the nucleic acid sequence comprises SEQ ID NO: 25 toencode amino acids 1-118 of human RAGE or a fragment thereof. Forexample, a sequence comprising nucleotides 1-348 of SEQ ID NO: 25 may beused to encode amino acids 1-116 of human RAGE. Or, the nucleic acid maycomprise SEQ ID NO: 26 to encode amino acids 1-123 of human RAGE. Or,the nucleic acid may comprise SEQ ID NO: 27 to encode amino acids 1-136of human RAGE. Or, the nucleic acid may comprise SEQ ID NO: 28 to encodeamino acids 1-230 of human RAGE. Or, the nucleic acid may comprise SEQID NO: 29 to encode amino acids 1-251 of human RAGE. Or fragments ofthese nucleic acid sequences may be used to encode RAGE polypeptidefragments.

The fusion protein may include several types of peptides that are notderived from RAGE or a fragment thereof. The second polypeptide of thefusion protein may comprise a polypeptide derived from animmunoglobulin. The heavy chain (or portion thereof) may be derived fromany one of the known heavy chain isotypes: IgG (γ), IgM (μ), IgD (δ),IgE (ε), or IgA (α). In addition, the heavy chain (or portion thereof)may be derived from any one of the known heavy chain subtypes: IgG1(γ1), IgG2 (γ2), IgG3 (γ3), IgG4 (γ4), IgA1 (α1), IgA2 (α2), ormutations of these isotypes or subtypes that alter the biologicalactivity. The second polypeptide may comprise the C_(H)2 and C_(H)3domains of a human IgG1 or a portion of either, or both, of thesedomains. As an example embodiments, the polypeptide comprising theC_(H)2 and C_(H)3 domains of a human IgG1 or a portion thereof maycomprise SEQ ID NO: 38 or SEQ ID NO: 40. The immunoglobulin peptide maybe encoded by the nucleic acid sequence of SEQ ID NO: 39 or SEQ ID NO:41.

The Fc portion of the immunoglobulin chain may be proinflammatory invivo. Thus, the RAGE fusion protein of the present invention maycomprise an interdomain linker derived from RAGE rather than aninterdomain hinge polypeptide derived from an immunoglobulin. Forexample, in one embodiment, the fusion protein may be encoded by arecombinant DNA construct. Also, the method may comprise the step ofincorporating the DNA construct into an expression vector. Also, themethod may comprise transfecting the expression vector into a host cell.

Thus, in one embodiment, the present invention comprises a method ofmaking a RAGE fusion protein comprising the step of covalently linking aRAGE polypeptide to a polypeptide comprising a C_(H)2 domain of animmunoglobulin or a portion of a C_(H)2 domain of an immunoglobulin. Inone embodiment, the fusion protein may comprise a RAGE ligand bindingsite. The RAGE ligand binding site may comprise the V domain of RAGE, ora portion thereof. In an embodiment, the RAGE ligand binding sitecomprises SEQ ID NO: 9 or a sequence 90% identical thereto, or SEQ IDNO: 10 or a sequence 90% identical thereto

For example, in one embodiment, the present invention comprises anucleic acid encoding a RAGE polypeptide directly linked to apolypeptide comprising a C_(H)2 domain of an immunoglobulin, or afragment thereof. In one embodiment, the C_(H)2 domain, or a fragmentthereof, comprises SEQ ID NO: 42. The second polypeptide may comprisethe C_(H)2 and C_(H)3 domains of a human IgG1. As an example embodiment,the polypeptide comprising the C_(H)2 and C_(H)3 domains of a human IgG1may comprise SEQ ID NO: 38 or SEQ ID NO: 40. The immunoglobulin peptidemay be encoded by the nucleic acid sequence of SEQ ID NO: 39 or SEQ IDNO: 41.

In one embodiment, the RAGE polypeptide may comprise a RAGE interdomainlinker linked to a RAGE immunoglobulin domain such that the C-terminalamino acid of the RAGE immunoglobulin domain is linked to the N-terminalamino acid of the interdomain linker, and the C-terminal amino acid ofthe RAGE interdomain linker is directly linked to the N-terminal aminoacid of a polypeptide comprising a C_(H)2 domain of an immunoglobulin,or a fragment thereof. The polypeptide comprising a C_(H)2 domain of animmunoglobulin may comprise a polypeptide comprising the C_(H)2 andC_(H)3 domains of a human IgG1 or a portion of both, or either, of thesedomains. As an example embodiment, the polypeptide comprising the C_(H)2and C_(H)3 domains of a human IgG1, or a portion thereof, may compriseSEQ ID NO: 38 or SEQ ID NO: 40.

The fusion protein of the present invention may comprise a single ormultiple domains from RAGE. Also, the RAGE polypeptide comprising aninterdomain linker linked to a RAGE immunoglobulin domain may comprise afragment of a full-length RAGE protein. For example, in one embodiment,the fusion protein may comprise two immunoglobulin domains derived fromRAGE protein and two immunoglobulin domains derived from a human Fcpolypeptide. The fusion protein may comprise a first RAGE immunoglobulindomain and a first interdomain linker linked to a second RAGEimmunoglobulin domain and a second RAGE interdomain linker, such thatthe N-terminal amino acid of the first interdomain linker is linked tothe C-terminal amino acid of the first RAGE immunoglobulin domain, theN-terminal amino acid of the second RAGE immunoglobulin domain is linkedto C-terminal amino acid of the first interdomain linker, the N-terminalamino acid of the second interdomain linker is linked to C-terminalamino acid of the RAGE second immunoglobulin domain, and the C-terminalamino acid of the RAGE second interdomain linker is directly linked tothe N-terminal amino acid of the polypeptide comprising a C_(H)2immunoglobulin domain or fragment thereof. For example, the RAGEpolypeptide may comprise amino acids 23-251 of human RAGE (SEQ ID NO:19) or a sequence 90% identical thereto, or amino acids 24-251 of humanRAGE (SEQ ID NO: 20) or a sequence 90% identical thereto, correspondingto the V-domain, the C1 domain, the interdomain linker linking these twodomains, and a second interdomain linker downstream of C1. In oneembodiment, a nucleic acid construct comprising SEQ ID NO: 30 or afragment thereof may encode for a four domain RAGE fusion protein.

Alternatively, a three domain fusion protein may comprise oneimmunoglobulin domain derived from RAGE and two immunoglobulin domainsderived from a human Fc polypeptide. For example, the fusion protein maycomprise a single RAGE immunoglobulin domain linked via a RAGEinterdomain linker to the N-terminal amino acid of the polypeptidecomprising a C_(H)2 immunoglobulin domain or a fragment thereof. Forexample, the RAGE polypeptide may comprise amino acids 23-136 of humanRAGE (SEQ ID NO: 15) or a sequence 90% identical thereto or amino acids24-136 of human RAGE (SEQ ID NO: 16) or a sequence 90% identical theretocorresponding to the V domain of RAGE and a downstream interdomainlinker. In one embodiment, a nucleic acid construct comprising SEQ IDNO: 31 or a fragment thereof may encode for a three domain RAGE fusionprotein.

A RAGE interdomain linker fragment may comprise a peptide sequence thatis naturally downstream of, and thus, linked to, a RAGE immunoglobulindomain. For example, for the RAGE V domain, the interdomain linker maycomprise amino acid sequences that are naturally downstream from the Vdomain. In an embodiment, the linker may comprise SEQ ID NO: 21,corresponding to amino acids 117-123 of full-length RAGE. Or, the linkermay comprise a peptide having additional portions of the natural RAGEsequence. For example, a interdomain linker comprising several aminoacids (e.g., 1-3, 1-5, or 1-10, or 1-15 amino acids) upstream anddownstream of SEQ ID NO: 21 may be used. Thus, in one embodiment, theinterdomain linker comprises SEQ ID NO: 23 comprising amino acids117-136 of full-length RAGE. Or, fragments of SEQ ID NO: 21 deleting,for example, 1, 2, or 3, amino acids from either end of the linker maybe used. In alternate embodiments, the linker may comprise a sequencethat is 70% identical, or 80% identical, or 90% identical to SEQ ID NO:21 or SEQ ID NO: 23.

For the RAGE C1 domain, the linker may comprise peptide sequence that isnaturally downstream of the C1 domain. In an embodiment, the linker maycomprise SEQ ID NO: 22, corresponding to amino acids 222-251 offull-length RAGE. Or, the linker may comprise a peptide havingadditional portions of the natural RAGE sequence. For example, a linkercomprising several (1-3, 1-5, or 1-10, or 1-15 amino acids) amino acidsupstream and downstream of SEQ ID NO: 22 may be used. Or, fragments ofSEQ ID NO: 22 may be used, deleting for example, 1-3, 1-5, or 1-10, or1-15 amino acids from either end of the linker. For example, in oneembodiment, a RAGE interdomain linker may comprise SEQ ID NO: 24,corresponding to amino acids 222-226. Or an interdomain linker maycomprise SEQ ID NO: 44, corresponding to RAGE amino acids 318-342.

The method may further comprise the step of incorporating the DNAconstruct into an expression vector. Thus, in a embodiment, the presentinvention comprises an expression vector that encodes for a fusionprotein comprising a RAGE polypeptide directly linked to a polypeptidecomprising a C_(H)2 domain of an immunoglobulin or a portion of a C_(H)2domain of an immunoglobulin. In an embodiment, the RAGE polypeptidecomprise constructs, such as those described herein, having a RAGEinterdomain linker linked to a RAGE immunoglobulin domain such that theC-terminal amino acid of the RAGE immunoglobulin domain is linked to theN-terminal amino acid of the interdomain linker, and the C-terminalamino acid of the RAGE interdomain linker is directly linked to theN-terminal amino acid of a polypeptide comprising a C_(H)2 domain of animmunoglobulin, or a portion thereof. For example, the expression vectorused to transfect the cells may comprise the nucleic acid sequence SEQID NO:30, or a fragment thereof, or SEQ ID NO: 31, or a fragmentthereof.

The method may further comprise the step of transfecting a cell with theexpression vector of the present invention. Thus, in an embodiment, thepresent invention comprises a cell transfected with the expressionvector that expressed the RAGE fusion protein of the present invention,such that the cell expresses a fusion protein comprising a RAGEpolypeptide directly linked to a polypeptide comprising a C_(H)2 domainof an immunoglobulin or a portion of a C_(H)2 domain of animmunoglobulin. In an embodiment, the RAGE polypeptide compriseconstructs, such as those described herein, having a RAGE interdomainlinker linked to a RAGE immunoglobulin domain such that the C-terminalamino acid of the RAGE immunoglobulin domain is linked to the N-terminalamino acid of the interdomain linker, and the C-terminal amino acid ofthe RAGE interdomain linker is directly linked to the N-terminal aminoacid of a polypeptide comprising a C_(H)2 domain of an immunoglobulin,or a portion thereof. For example, the expression vector may comprisethe nucleic acid sequence SEQ ID NO:30, or a fragment thereof, or SEQ IDNO: 31, or a fragment thereof.

For example, plasmids may be constructed to express RAGE-IgG Fc fusionproteins by fusing different lengths of a 5′ cDNA sequence of human RAGEwith a 3′ cDNA sequence of human IgG1 Fc (γ1). The expression cassettesequences may be inserted into an expression vector such as pcDNA3.1expression vector (Invitrogen, Calif.) using standard recombinanttechniques.

Also, the method may comprise transfecting the expression vector into ahost cell. In one embodiment, the recombinant may be transfected intoChinese Hamster Ovary cells and expression optimized. In alternateembodiments, the cells may produce 0.1 to 20 grams/liter, or 0.5 to 10grams/liter, or about 1-2 grams/liter.

As is known in the art, such nucleic acid constructs may be modified bymutation, as for example, by PCR amplification of a nucleic acidtemplate with primers comprising the mutation of interest. In this way,polypeptides comprising varying affinity for RAGE ligands may bedesigned. In one embodiment, the mutated sequences may be 90% or moreidentical to the starting DNA. As such, variants may include nucleotidesequences that hybridize under stringent conditions (i.e., equivalent toabout 20-27° C. below the melting temperature (TM) of the DNA duplex in1 molar salt).

The coding sequence may be expressed by transfecting the expressionvector into an appropriate host. For example, the recombinant vectorsmay be stably transfected into Chinese Hamster Ovary (CHO) cells, andcells expressing the fusion protein selected and cloned. In anembodiment, cells expressing the recombinant construct are selected forplasmid-encoded neomycin resistance by applying antibiotic G418.Individual clones may be selected and clones expressing high levels ofrecombinant protein as detected by Western Blot analysis of the cellsupernatant may be expanded, and the gene product purified by affinitychromatography using Protein A columns.

Sample embodiments of recombinant nucleic acids that encode the fusionproteins of the present invention are shown in FIGS. 2-5. For example,as described above, the fusion protein produced by the recombinant DNAconstruct may comprise a RAGE polypeptide linked to a second, non-RAGEpolypeptide. The fusion protein may comprise two domains derived fromRAGE protein and two domains derived from an immunoglobulin. An examplenucleic acid construct encoding a fusion protein, TTP-4000 (TT4), havingthis type of structure is shown as FIG. 2 (SEQ ID NO: 30). As shown inFIG. 2, coding sequence 1-753 (highlighted in bold) encodes the RAGEN-terminal protein sequence whereas the sequence from 754-1386 encodesthe IgG Fc protein sequence.

When derived from SEQ ID NO: 30, or a sequence 90% identical thereto,the fusion protein may comprise the four domain amino acid sequence ofSEQ ID NO: 32, or the polypeptide with the signal sequence removed(e.g., SEQ ID NO: 33 or SEQ ID NO: 34) (FIG. 4). In FIG. 4, the RAGEamino acid sequence is highlighted with bold font. The immunoglobulinsequence is the C_(H)2 and C_(H)3 immunoglobulin domains of IgG. Asshown in FIG. 6B, the first 251 amino acids of the full-length TTP-4000RAGE fusion protein contains as the RAGE polypeptide sequence a signalsequence comprising amino acids 1-22/23, the V immunoglobulin domain(including the ligand binding site) comprising amino acids 23/24-116, aninterdomain linker comprising amino acids 117 to 123, a secondimmunoglobulin domain (C1) comprising amino acids 124-221, and adownstream interdomain linker comprising amino acids 222-251.

In an embodiment, the fusion protein may not necessarily comprise thesecond RAGE immunoglobulin domain. For example, the fusion protein maycomprise one immunoglobulin domain derived from RAGE and twoimmunoglobulin domains derived from a human Fc polypeptide. An examplenucleic acid construct encoding this type of fusion protein is shown asFIG. 3 (SEQ ID NO: 31). As shown in FIG. 3, the coding sequence fromnucleotides 1 to 408 (highlighted in bold) encodes the RAGE N-terminalprotein sequence, whereas the sequence from 409-1041 codes the IgG1 Fc(γ1) protein sequence.

When derived from SEQ ID NO: 31, or a sequence 90% identical thereto,the fusion protein may comprise the three domain amino acid sequence ofSEQ ID NO: 35, or the polypeptide with the signal sequence removed(e.g., SEQ ID NO: 36 or SEQ ID NO: 37) (FIG. 5). In FIG. 5, the RAGEamino acid sequence is highlighted with bold font. As shown in FIG. 6B,the first 136 amino acids of the full-length TTP-3000 RAGE fusionprotein contains as the RAGE polypeptide a signal sequence comprisingamino acids 1-22/23, the V immunoglobulin domain (including the ligandbinding site) comprising amino acids 23/24-116, and an interdomainlinker comprising amino acids 117 to 136. The sequence from 137 to 346includes the C_(H)2 and C_(H)3 immunoglobulin domains of IgG.

The fusion proteins of the present invention may comprise improved invivo stability over RAGE polypeptides not comprising a secondpolypeptide. The fusion protein may be further modified to increasestability, efficacy, potency and bioavailability. Thus, the fusionproteins of the present invention may be modified by post-translationalprocessing or by chemical modification. For example, the fusion proteinmay be synthetically prepared to include L-, D-, or unnatural aminoacids, alpha-disubstituted amino acids, or N-alkyl amino acids.Additionally, proteins may be modified by acetylation, acylation,ADP-ribosylation, amidation, attachment of lipids such asphosphatidyinositol, formation of disulfide bonds, and the like.Furthermore, polyethylene glycol can be added to increase the biologicalstability of the fusion protein.

Binding of RAGE Antagonists to RAGE Fusion Proteins

The fusion proteins of the present invention may comprise a number ofapplications. For example, the fusion protein of the present inventionmay be used in a binding assay to identify RAGE ligands, such as RAGEagonists, antagonists, or modulators.

For example, in one embodiment, the present invention provides a methodfor detection of RAGE modulators comprising: (a) providing a fusionprotein comprising a RAGE polypeptide linked to a second, non-RAGEpolypeptide, where the RAGE polypeptide comprises a ligand binding site;(b) mixing a compound of interest and a ligand having a known bindingaffinity for RAGE with the fusion protein; and (c) measuring binding ofthe known RAGE ligand to the RAGE fusion protein in the presence of thecompound of interest. In an embodiment, the ligand binding sitecomprises the most N-terminal domain of the fusion protein.

The RAGE fusion proteins may also provide kits for the detection of RAGEmodulators. For example, in one embodiment, a kit of the presentinvention may comprise (a) a compound having known binding affinity toRAGE as a positive control; (b) a RAGE fusion protein comprising a RAGEpolypeptide linked to a second, non-RAGE polypeptide, wherein the RAGEpolypeptide comprises a RAGE ligand binding site; and (c) instructionsfor use. In an embodiment, the ligand binding site comprises the mostN-terminal domain of the fusion protein.

The RAGE protein or polypeptide may comprise full-length human RAGE(e.g., SEQ ID NO: 1), or a fragment of human RAGE. In an embodiment, theRAGE polypeptide does not include any signal sequence residues. Thesignal sequence of RAGE may comprise either residues 1-22 or residues1-23 of full length RAGE (SEQ ID NO: 1). In alternate embodiments, theRAGE polypeptide may comprise a sequence 70%, 80%, or 90% identical tohuman RAGE, or a fragment thereof. For example, in one embodiment, theRAGE polypeptide may comprise human RAGE, or a fragment thereof, withGlycine as the first residue rather than a Methionine (see e.g., Neeperet al., (1992)). Or, the human RAGE may comprise full-length RAGE withthe signal sequence removed (e.g., SEQ ID NO: 2 or SEQ ID NO: 3) (FIGS.1A and 1B) or a portion of that amino acid sequence. The fusion proteinsof the present invention may also comprise sRAGE (e.g., SEQ ID NO: 4), apolypeptide 90% identical to sRAGE, or a fragment of sRAGE. For example,the RAGE polypeptide may comprise human sRAGE, or a fragment thereof,with Glycine as the first residue rather than a Methionine (see e.g.,Neeper et al., (1992)). Or, the human RAGE may comprise sRAGE with thesignal sequence removed (e.g., SEQ ID NO: 5 or SEQ ID NO: 6) (FIG. 1C)or a portion of that amino acid sequence. In other embodiments, the RAGEprotein may comprise a V domain (e.g., SEQ ID NO: 7 or SEQ ID NO: 8;FIG. 1D). Or, a sequence 90% identical to the V domain or a fragmentthereof may be used. Or, the RAGE protein may comprise a fragment ofRAGE comprising a portion of the V domain (e.g., SEQ ID NO: 9 or SEQ IDNO: 10, FIG. 1D). In an embodiment, the ligand binding site may compriseSEQ ID NO: 9, or a sequence 90% identical thereto, or SEQ ID NO: 10, ora sequence 90% identical thereto. In yet another embodiment, the RAGEfragment is a synthetic peptide.

The fusion protein may include several types of peptides that are notderived from RAGE or a fragment thereof. The second polypeptide of thefusion protein may comprise a polypeptide derived from animmunoglobulin. The heavy chain (or portion thereof) may be derived fromany one of the known heavy chain isotypes: IgG (γ), IgM (μ), IgD (δ),IgE (ε), or IgA (α). In addition, the heavy chain (or portion thereof)may be derived from any one of the known heavy chain subtypes: IgG1(γ1), IgG2 (γ2), IgG3 (γ3), IgG4 (γ4), IgA1 (α1), IgA2 (α2), ormutations of these isotypes or subtypes that alter the biologicalactivity. The second polypeptide may comprise the C_(H)2 and C_(H)3domains of a human IgG1 or a portion of either, or both, of thesedomains. As an example embodiments, the polypeptide comprising theC_(H)2 and C_(H)3 domains of a human IgG1 or a portion thereof maycomprise SEQ ID NO: 38 or SEQ ID NO: 40. The immunoglobulin peptide maybe encoded by the nucleic acid sequence of SEQ ID NO: 39 or SEQ ID NO:41.

The Fc portion of the immunoglobulin chain may be proinflammatory invivo. Thus, the RAGE fusion protein of the present invention maycomprise an Fc sequence derived from RAGE rather than an immunoglobulinchain. In an embodiment, the fusion protein may comprise a RAGEimmunoglobulin domain linked to a polypeptide comprising a C_(H)2immunoglobulin domain or a fragment thereof. In one embodiment, the RAGEpolypeptide may comprise a RAGE interdomain linker linked to a RAGEimmunoglobulin domain such that the C-terminal amino acid of the RAGEimmunoglobulin domain is linked to the N-terminal amino acid of theinterdomain linker, and the C-terminal amino acid of the RAGEinterdomain linker is directly linked to the N-terminal amino acid of apolypeptide comprising a C_(H)2 domain of an immunoglobulin, or afragment thereof. The polypeptide comprising a C_(H)2 domain of animmunoglobulin may comprise a polypeptide comprising the C_(H)2 andC_(H)3 domains of a human IgG1 or a portion of both, or either, of thesedomains. As an example embodiment, the polypeptide comprising the C_(H)2and C_(H)3 domains of a human IgG1, or a portion thereof, may compriseSEQ ID NO: 38 or SEQ ID NO: 40.

The fusion protein of the present invention may comprise a single ormultiple domains from RAGE. Also, the RAGE polypeptide comprising aninterdomain linker linked to a RAGE immunoglobulin domain may comprise afragment of a full-length RAGE protein. For example, in one embodiment,the fusion protein may comprise two immunoglobulin domains derived fromRAGE protein and two immunoglobulin domains derived from a human Fcpolypeptide. The fusion protein may comprise a first RAGE immunoglobulindomain and a first interdomain linker linked to a second RAGEimmunoglobulin domain and a second RAGE interdomain linker, such thatthe N-terminal amino acid of the first interdomain linker is linked tothe C-terminal amino acid of the first RAGE immunoglobulin domain, theN-terminal amino acid of the second RAGE immunoglobulin domain is linkedto C-terminal amino acid of the first interdomain linker, the N-terminalamino acid of the second interdomain linker is linked to C-terminalamino acid of the RAGE second immunoglobulin domain, and the C-terminalamino acid of the RAGE second interdomain linker is directly linked tothe N-terminal amino acid of the polypeptide comprising a C_(H)2immunoglobulin domain or fragment thereof. For example, the RAGEpolypeptide may comprise amino acids 23-251 of human RAGE (SEQ ID NO:19) or a sequence 90% identical thereto, or amino acids 24-251 of humanRAGE (SEQ ID NO: 20) or a sequence 90% identical thereto, correspondingto the V-domain, the C1 domain, the interdomain linker linking these twodomains, and a second interdomain linker downstream of C1. In oneembodiment, a nucleic acid construct comprising SEQ ID NO: 30 or afragment thereof may encode for a four domain RAGE fusion protein.

Alternatively, a three domain fusion protein may comprise oneimmunoglobulin domain derived from RAGE and two immunoglobulin domainsderived from a human Fc polypeptide. For example, the fusion protein maycomprise a single RAGE immunoglobulin domain linked via a RAGEinterdomain linker to the N-terminal amino acid of the polypeptidecomprising a C_(H)2 immunoglobulin domain or a fragment thereof. Forexample, the RAGE polypeptide may comprise amino acids 23-136 of humanRAGE (SEQ ID NO: 15) or a sequence 90% identical thereto or amino acids24-136 of human RAGE (SEQ ID NO: 16) or a sequence 90% identical theretocorresponding to the V domain of RAGE and a downstream interdomainlinker. In one embodiment, a nucleic acid construct comprising SEQ IDNO: 31 or a fragment thereof may encode for a three domain RAGE fusionprotein.

As described herein, RAGE interdomain linker fragment may comprise apeptide sequence that is naturally downstream of, and thus, linked to, aRAGE immunoglobulin domain. For example, for the RAGE V domain, theinterdomain linker may comprise amino acid sequences that are naturallydownstream from the V domain. In an embodiment, the linker may compriseSEQ ID NO: 21, corresponding to amino acids 117-123 of full-length RAGE.Or, the linker may comprise a peptide having additional portions of thenatural RAGE sequence. For example, a interdomain linker comprisingseveral amino acids (e.g., 1-3, 1-5, or 1-10, or 1-15 amino acids)upstream and downstream of SEQ ID NO: 21 may be used. Thus, in oneembodiment, the interdomain linker comprises SEQ ID NO: 23 comprisingamino acids 117-136 of full-length RAGE. Or, fragments of SEQ ID NO: 21deleting, for example, 1, 2, or 3, amino acids from either end of thelinker may be used. In alternate embodiments, the linker may comprise asequence that is 70% identical, or 80% identical, or 90% identical toSEQ ID NO: 21 or SEQ ID NO: 23.

For the RAGE C1 domain, the linker may comprise peptide sequence that isnaturally downstream of the C1 domain. In an embodiment, the linker maycomprise SEQ ID NO: 22, corresponding to amino acids 222-251 offull-length RAGE. Or, the linker may comprise a peptide havingadditional portions of the natural RAGE sequence. For example, a linkercomprising several (1-3, 1-5, or 1-10, or 1-15 amino acids) amino acidsupstream and downstream of SEQ ID NO: 22 may be used. Or, fragments ofSEQ ID NO: 22 may be used, deleting for example, 1-3, 1-5, or 1-10, or1-15 amino acids from either end of the linker. For example, in oneembodiment, a RAGE interdomain linker may comprise SEQ ID NO: 24,corresponding to amino acids 222-226. Or an interdomain linker maycomprise SEQ ID NO: 44, corresponding to RAGE amino acids 318-342.

For example, the RAGE fusion protein may be used in a binding assay toidentify potential RAGE ligands. In one example embodiment of such abinding assay, a known RAGE ligand may coated onto a solid substrate(e.g., Maxisorb plates) at a concentration of about 5 micrograms perwell, where each well contains a total volume of about 100 microliters(μL). The plates may be incubated at 4° C. overnight to allow the ligandto absorb. Alternatively, shorter incubation periods at highertemperature (e.g., room temperature) may be used. After a period of timeto allow for the ligand to bind to the substrate, the assay wells may beaspirated and a blocking buffer (e.g., 1% BSA in 50 mM imidizole buffer,pH 7.2) may be added to block nonspecific binding. For example, blockingbuffer may be added to the plates for 1 hour at room temperature. Theplates may then be aspirated and/or washed with a wash buffer. In oneembodiment, a buffer comprising 20 mM Imidizole, 150 mM NaCl, 0.05%Tween-20, 5 mM CaCl₂ and 5 mM MgCl₂, pH 7.2 may be used as a washbuffer. The fusion protein may then added at increasing dilutions to theassay wells. The RAGE fusion protein may then be allowed to incubatewith the immobilized ligand in the assay well such that binding canattain equilibrium. In one embodiment, the RAGE fusion protein isallowed to incubate with the immobilized ligand for about one hour at37° C. In alternate embodiments, longer incubation periods at lowertemperatures may be used. After the fusion protein and immobilizedligand have been incubated, the plate may be washed to remove anyunbound fusion protein. The fusion protein bound to the immobilizedligand may be detected in a variety of ways. In one embodiment,detection employs an ELISA. Thus, in one embodiment, an immunodetectioncomplex containing a monoclonal mouse anti-human IgG1, biotinylated goatanti-mouse IgG, and an avidin linked alkaline phosphatase may be addedto the fusion protein immobilized in the assay well. The immunodetectioncomplex may be allowed to bind to the immobilized fusion protein suchthat binding between the fusion protein and the immunodetection complexattains equilibrium. For example, the complex may be allowed to bind tothe fusion protein for one hour at room temperature. At that point, anyunbound complex may be removed by washing the assay well with washbuffer. The bound complex may be detected by adding the alkalinephosphatase substrate, para-nitrophenylphosphate (PNPP), and measuringconversion of PNPP to para-nitrophenol (PNP) as an increase inabsorbance at 405 nm.

In an embodiment, RAGE ligand bind to the RAGE fusion protein withnanomolar (nM) or micromolar (μM) affinity. An experiment illustratingbinding of RAGE ligands to RAGE fusion proteins of the present inventionis shown in FIG. 7. Solutions of TTP-3000 (TT3) and TTP-4000 (TT4)having initial concentrations of 1.082 mg/mL, and 370 μg/mL,respectively, were prepared. As shown FIG. 7, at various dilutions, thefusion proteins TTP-3000 and TTP-4000 are able to bind to immobilizedRAGE ligands Amyloid-beta (Abeta) (Amyloid Beta (1-40) from Biosource),S100b (S100), and amphoterin (Ampho), resulting in an increase inabsorbance. In the absence of ligand (i.e., coating with only BSA) therewas no increase in absorbance.

The binding assay of the present invention may be used to quantifyligand binding to RAGE. In alternate embodiments, RAGE ligands may bindto the fusion protein of the present invention with binding affinitiesranging from 0.1 to 1000 nanomolar (nM), or from 1 to 500 nM, or from 10to 80 nM.

The fusion protein of the present invention may also be used to identifycompounds having the ability to bind to RAGE. As shown in FIGS. 8 and 9,respectively, a RAGE ligand may be assayed for its ability to competewith immobilized amyloid beta for binding to TTP-4000 (TT4) or TTP-3000(TT3) fusion proteins. Thus, it may be seen that a RAGE ligand at afinal assay concentration (FAC) of 10 μM can displace binding of RAGEfusion protein to amyloid-beta at concentrations of 1:3, 1:10, 1:30, and1:100 of the initial TTP-4000 solution (FIG. 8) or TTP-3000 (FIG. 9).

Modulation of Cellular Effectors

Embodiments of the fusion proteins of the present invention may be usedto modulate a biological response mediated by RAGE. For example, thefusion proteins may be designed to modulate RAGE-induced increases ingene expression. Thus, in an embodiment, fusion proteins of the presentinvention may be used to modulate the function of biological enzymes.For example, the interaction between RAGE and its ligands may generateoxidative stress and activation of NF-κB, and NF-κB regulated genes,such as the cytokines IL-1β, TNF-α, and the like. In addition, severalother regulatory pathways, such as those involving p21ras, MAP kinases,ERK1, and ERK2, have been shown to be activated by binding of AGEs andother ligands to RAGE.

Use of the fusion proteins of the present invention to modulateexpression of the cellular effector TNF-α is shown in FIG. 10. THP-1myeloid cells may be cultured in RPMI-1640 media supplemented with 10%FBS and induced to secrete TNF-α via stimulation of RAGE with S100b.When such stimulation occurs in the presence of a RAGE fusion protein,induction of TNF-α by S100b binding to RAGE may be inhibited. Thus, asshown in FIG. 10, addition of 10 μg TTP-3000 (TT3) or TTP-4000 (TT4)RAGE fusion protein reduces S100b induction of TNF-α by about 50% to75%. Fusion protein TTP-4000 may be at least as effective in blockingS100b induction of TNF-α as is sRAGE (FIG. 10). Specificity of theinhibition for the RAGE sequences of TTP-4000 and TTP-3000 is shown bythe experiment in which IgG alone was added to S100b stimulated cells.Addition of IgG and S100b to the assay shows the same levels of TNF-α asS100b alone.

Physiological Characteristics of RAGE Fusion Proteins

While sRAGE can have a therapeutic benefit in the modulation ofRAGE-mediated diseases, human sRAGE may have limitations as astand-alone therapeutic based on the relatively short half-life of sRAGEin plasma. For example, whereas rodent sRAGE has a half-life in normaland diabetic rats of approximately 20 hours, human sRAGE has a half-lifeof less than 2 hours when assessed by retention of immunoreactivitysRAGE (Renard et al., J. Pharmacol. Exp. Ther., 290:1458-1466 (1999)).

To generate a RAGE therapeutic that has similar binding characteristicsas sRAGE, but a more stable pharmacokinetic profile, a RAGE fusionprotein comprising a RAGE ligand binding site linked to one or morehuman immunoglobulin domains may be used. As is known in the art, theimmunoglobulin domains may include the Fc portion of the immunoglobulinheavy chain.

The immunoglobulin Fc portion may confer several attributes to a fusionprotein. For example, the Fc fusion protein may increase the serumhalf-life of such fusion proteins, often from hours to several days. Theincrease in pharmacokinetic stability is generally a result of theinteraction of the linker between C_(H)2 and C_(H)3 regions of the Fcfragment with the FcRn receptor (Wines et al., J. Immunol.,164:5313-5318 (2000)).

Although fusion proteins comprising an immunoglobulin Fc polypeptide mayprovide the advantage of increased stability, immunoglobulin fusionproteins may elicit an inflammatory response when introduced into ahost. The inflammatory response may be due, in large part, to the Fcportion of the immunoglobulin of the fusion protein. The proinflammatoryresponse may be a desirable feature if the target is expressed on adiseased cell type that needs to be eliminated (e.g., a cancer cell, anor a population of lymphocytes causing an autoimmune disease). Theproinflammatory response may be a neutral feature if the target is asoluble protein, as most soluble proteins do not activateimmunoglobulins. However, the proinflammatory response may be a negativefeature if the target is expressed on cell types whose destruction wouldlead to untoward side-effects. Also, the proinflammatory response may bea negative feature if an inflammatory cascade is established at the siteof a fusion protein binding to a tissue target, since many mediators ofinflammation may be detrimental to surrounding tissue, and/or may causesystemic effects.

The primary proinflammatory site on immunoglobulin Fc fragments resideson the hinge region between the C_(H)1 and C_(H)2. This hinge regioninteracts with the FcR1-3 on various leukocytes and trigger these cellsto attack the target. (Wines et al., J. Immunol., 164:5313-5318 (2000)).

As therapeutics for RAGE-mediated diseases, RAGE fusion proteins may notrequire the generation of an inflammatory response. Thus, embodiments ofthe RAGE fusion proteins of the present invention may comprise a fusionprotein comprising a RAGE polypeptide linked to an immunoglobulindomain(s) where the Fc hinge region from the immunoglobulin is removedand replaced with a RAGE polypeptide. In this way, interaction betweenthe RAGE fusion protein and Fc receptors on inflammatory cells may beminimized. It may be important, however, to maintain proper stacking andother three-dimensional structural interactions between the variousimmunoglobulin domains of the fusion protein. Thus, embodiments of thefusion proteins of the present invention may substitute the biologicallyinert, but structurally similar RAGE interdomain linker that separatesthe V and C1 domains of RAGE, or the linker that separates the C1 and C2domains of RAGE, in lieu of the normal hinge region of theimmunoglobulin heavy chain. Thus, the RAGE polypeptide of the fusionprotein may comprise an interdomain linker sequence that is naturallyfound downstream of a RAGE immunoglobulin domain to form a RAGEimmunglobulin domain/linker fragment. In this way, the three dimensionalinteractions between the immunoglobulin domains contributed by eitherRAGE or the immunoglobulin may be maintained.

In an embodiment, a RAGE fusion protein of the present invention maycomprise a substantial increase in pharmacokinetic stability as comparedto sRAGE. For example, FIG. 11 shows that once the RAGE fusion proteinTTP-4000 has saturated its ligands, it may retain a half-life of greaterthan 300 hours. This may be contrasted with the half-life for sRAGE ofonly a few hours in human plasma.

Thus, in an embodiment, the RAGE fusion proteins of the presentinvention may be used to antagonize binding of physiological ligands toRAGE as a means to treat RAGE-mediated diseases without generating anunacceptable amount of inflammation. The fusion proteins of the presentinvention may exhibit a substantial decrease in generating aproinflammatory response as compared to IgG. For example, as shown inFIG. 12, the RAGE fusion protein TTP-4000 does not stimulate TNF-αrelease from cells under conditions where human IgG stimulation of TNF-αrelease is detected.

Treatment of Disease with RAGE Fusion Proteins

The present invention may also comprise methods for the treatment ofRAGE-mediated disorder in a human subject. In an embodiment, the methodmay comprise administering to a subject a fusion protein comprising aRAGE polypeptide comprising a RAGE ligand binding site linked to asecond, non-RAGE polypeptide. In one embodiment, the fusion protein maycomprise a RAGE ligand binding site. In an embodiment, the ligandbinding site comprises the most N-terminal domain of the fusion protein.The RAGE ligand binding site may comprise the V domain of RAGE, or aportion thereof. In an embodiment, the RAGE ligand binding sitecomprises SEQ ID NO: 9 or a sequence 90% identical thereto, or SEQ IDNO: 10 or a sequence 90% identical thereto.

In an embodiment, the RAGE polypeptide may be linked to a polypeptidecomprising an immunoglobulin domain or a portion (e.g., a fragmentthereof) of an immunoglobulin domain. In one embodiment, the polypeptidecomprising an immunoglobulin domain comprises at least a portion of atleast one of the C_(H)2 or the C_(H)3 domains of a human IgG.

The RAGE protein or polypeptide may comprise full-length human RAGE(e.g., SEQ ID NO: 1), or a fragment of human RAGE. In an embodiment, theRAGE polypeptide does not include any signal sequence residues. Thesignal sequence of RAGE may comprise either residues 1-22 or residues1-23 of full length RAGE (SEQ ID NO: 1). In alternate embodiments, theRAGE polypeptide may comprise a sequence that is 70%, 80% or 90%identical to human RAGE, or a fragment thereof. For example, in oneembodiment, the RAGE polypeptide may comprise human RAGE, or a fragmentthereof, with Glycine as the first residue rather than a Methionine (seee.g., Neeper et al., (1992)). Or, the human RAGE may comprisefull-length RAGE with the signal sequence removed (e.g., SEQ ID NO: 2 orSEQ ID NO: 3) (FIGS. 1A and 1B) or a portion of that amino acidsequence. The fusion proteins of the present invention may also comprisesRAGE (e.g., SEQ ID NO: 4), a polypeptide 90% identical to sRAGE, or afragment of sRAGE. For example, the RAGE polypeptide may comprise humansRAGE, or a fragment thereof, with Glycine as the first residue ratherthan a Methionine (see e.g., Neeper et al., (1992)). Or, the human RAGEmay comprise sRAGE with the signal sequence removed (e.g., SEQ ID NO: 5or SEQ ID NO: 6) (FIG. 1C) or a portion of that amino acid sequence. Inother embodiments, the RAGE protein may comprise a V domain (e.g., SEQID NO: 7 or SEQ ID NO: 8; FIG. 1D). Or, a sequence 90% identical to theV domain or a fragment thereof may be used. Or, the RAGE protein maycomprise a fragment of RAGE comprising a portion of the V domain (e.g.,SEQ ID NO: 9 or SEQ ID NO: 10, FIG. 1D). In an embodiment, the ligandbinding site may comprise SEQ ID NO: 9, or a sequence 90% identicalthereto, or SEQ ID NO: 10, or a sequence 90% identical thereto. In yetanother embodiment, the RAGE fragment is a synthetic peptide.

The fusion protein may include several types of peptides that are notderived from RAGE or a fragment thereof. The second polypeptide of thefusion protein may comprise a polypeptide derived from animmunoglobulin. The heavy chain (or portion thereof) may be derived fromany one of the known heavy chain isotypes: IgG (γ), IgM (μ), IgD (δ),IgE (ε), or IgA (α). In addition, the heavy chain (or portion thereof)may be derived from any one of the known heavy chain subtypes: IgG1(γ1), IgG2 (γ2), IgG3 (γ3), IgG4 (γ4), IgA1 (α1), IgA2 (α2), ormutations of these isotypes or subtypes that alter the biologicalactivity. The second polypeptide may comprise the C_(H)2 and C_(H)3domains of a human IgG1 or a portion of either, or both, of thesedomains. As an example embodiments, the polypeptide comprising theC_(H)2 and C_(H)3 domains of a human IgG1 or a portion thereof maycomprise SEQ ID NO: 38 or SEQ ID NO: 40. The immunoglobulin peptide maybe encoded by the nucleic acid sequence of SEQ ID NO: 39 or SEQ ID NO:41.

For example, the RAGE polypeptide may comprise amino acids 23-116 ofhuman RAGE (SEQ ID NO: 7) or a sequence 90% identical thereto, or aminoacids 24-116 of human RAGE (SEQ ID NO: 8) or a sequence 90% identicalthereto, corresponding to the V domain of RAGE. Or, the RAGE polypeptidemay comprise amino acids 124-221 of human RAGE (SEQ ID NO: 11) or asequence 90% identical thereto, corresponding to the C1 domain of RAGE.In another embodiment, the RAGE polypeptide may comprise amino acids227-317 of human RAGE (SEQ ID NO: 12) or a sequence 90% identicalthereto, corresponding to the C2 domain of RAGE. Or, the RAGEpolypeptide may comprise amino acids 23-123 of human RAGE (SEQ ID NO:13) or a sequence 90% identical thereto, or amino acids 24-123 of humanRAGE (SEQ ID NO: 14) or a sequence 90% identical thereto, correspondingto the V domain of RAGE and a downstream interdomain linker. Or, theRAGE polypeptide may comprise amino acids 23-226 of human RAGE (SEQ IDNO: 17) or a sequence 90% identical thereto, or amino acids 24-226 ofhuman RAGE (SEQ ID NO: 18) or a sequence 90% identical thereto,corresponding to the V-domain, the C1 domain and the interdomain linkerlinking these two domains. Or, the RAGE polypeptide may comprise aminoacids 23-339 of human RAGE (SEQ ID NO: 5) or a sequence 90% identicalthereto, or 24-339 of human RAGE (SEQ ID NO: 6) or a sequence 90%identical thereto, corresponding to sRAGE (i.e., encoding the V, C1, andC2 domains and interdomain linkers). Or, fragments of each of thesesequences may be used.

The Fc portion of the immunoglobulin chain may be proinflammatory invivo. Thus, in one embodiment, the RAGE fusion protein of the presentinvention comprises an interdomain linker derived from RAGE rather thanan interdomain hinge polypeptide derived from an immunoglobulin.

Thus in one embodiment, the fusion protein may further comprise a RAGEpolypeptide directly linked to a polypeptide comprising a C_(H)2 domainof an immunoglobulin, or a fragment thereof. In one embodiment, theC_(H)2 domain, or a fragment thereof comprises SEQ ID NO: 42.

In one embodiment, the RAGE polypeptide comprises a RAGE interdomainlinker linked to a RAGE immunoglobulin domain such that the C-terminalamino acid of the RAGE immunoglobulin domain is linked to the N-terminalamino acid of the interdomain linker, and the C-terminal amino acid ofthe RAGE interdomain linker is directly linked to the N-terminal aminoacid of a polypeptide comprising a C_(H)2 domain of an immunoglobulin,or a fragment thereof. The polypeptide comprising a C_(H)2 domain of animmunoglobulin may comprise the C_(H)2 and C_(H)3 domains of a humanIgG1. As an example embodiment, the polypeptide comprising the C_(H)2and C_(H)3 domains of a human IgG1 may comprise SEQ ID NO: 38 or SEQ IDNO: 40.

The fusion protein of the present invention may comprise a single ormultiple domains from RAGE. Also, the RAGE polypeptide comprising aninterdomain linker linked to a RAGE immunoglobulin domain may comprise afragment of a full-length RAGE protein. For example, in one embodiment,the fusion protein may comprise two immunoglobulin domains derived fromRAGE protein and two immunoglobulin domains derived from a human Fcpolypeptide. The fusion protein may comprise a first RAGE immunoglobulindomain and a first interdomain linker linked to a second RAGEimmunoglobulin domain and a second RAGE interdomain linker, such thatthe N-terminal amino acid of the first interdomain linker is linked tothe C-terminal amino acid of the first RAGE immunoglobulin domain, theN-terminal amino acid of the second RAGE immunoglobulin domain is linkedto C-terminal amino acid of the first interdomain linker, the N-terminalamino acid of the second interdomain linker is linked to C-terminalamino acid of the RAGE second immunoglobulin domain, and the C-terminalamino acid of the RAGE second interdomain linker is directly linked tothe N-terminal amino acid of the polypeptide comprising a C_(H)2immunoglobulin domain or fragment thereof. For example, the RAGEpolypeptide may comprise amino acids 23-251 of human RAGE (SEQ ID NO:19) or a sequence 90% identical thereto, or amino acids 24-251 of humanRAGE (SEQ ID NO: 20) or a sequence 90% identical thereto, correspondingto the V-domain, the C1 domain, the interdomain linker linking these twodomains, and a second interdomain linker downstream of C1. In oneembodiment, a nucleic acid construct comprising SEQ ID NO: 30 or afragment thereof may encode for a four domain RAGE fusion protein.

Alternatively, a three domain fusion protein may comprise oneimmunoglobulin domain derived from RAGE and two immunoglobulin domainsderived from a human Fc polypeptide. For example, the fusion protein maycomprise a single RAGE immunoglobulin domain linked via a RAGEinterdomain linker to the N-terminal amino acid of the polypeptidecomprising a C_(H)2 immunoglobulin domain or a fragment thereof. Forexample, the RAGE polypeptide may comprise amino acids 23-136 of humanRAGE (SEQ ID NO: 15) or a sequence 90% identical thereto or amino acids24-136 of human RAGE (SEQ ID NO: 16) or a sequence 90% identical theretocorresponding to the V domain of RAGE and a downstream interdomainlinker. In one embodiment, a nucleic acid construct comprising SEQ IDNO: 31 or a fragment thereof may encode for a three domain RAGE fusionprotein.

A RAGE interdomain linker fragment may comprise a peptide sequence thatis naturally downstream of, and thus, linked to, a RAGE immunoglobulindomain. For example, for the RAGE V domain, the interdomain linker maycomprise amino acid sequences that are naturally downstream from the Vdomain. In an embodiment, the linker may comprise SEQ ID NO: 21,corresponding to amino acids 117-123 of full-length RAGE. Or, the linkermay comprise a peptide having additional portions of the natural RAGEsequence. For example, a interdomain linker comprising several aminoacids (e.g., 1-3, 1-5, or 1-10, or 1-15 amino acids) upstream anddownstream of SEQ ID NO: 21 may be used. Thus, in one embodiment, theinterdomain linker comprises SEQ ID NO: 23 comprising amino acids117-136 of full-length RAGE. Or, fragments of SEQ ID NO: 21 deleting,for example, 1, 2, or 3, amino acids from either end of the linker maybe used. In alternate embodiments, the linker may comprise a sequencethat is 70% identical, or 80% identical, or 90% identical to SEQ ID NO:21 or SEQ ID NO: 23.

For the RAGE C1 domain, the linker may comprise peptide sequence that isnaturally downstream of the C1 domain. In an embodiment, the linker maycomprise SEQ ID NO: 22, corresponding to amino acids 222-251 offull-length RAGE. Or, the linker may comprise a peptide havingadditional portions of the natural RAGE sequence. For example, a linkercomprising several (1-3, 1-5, or 1-10, or 1-15 amino acids) amino acidsupstream and downstream of SEQ ID NO: 22 may be used. Or, fragments ofSEQ ID NO: 22 may be used, deleting for example, 1-3, 1-5, or 1-10, or1-15 amino acids from either end of the linker. For example, in oneembodiment, a RAGE interdomain linker may comprise SEQ ID NO: 24,corresponding to amino acids 222-226. Or an interdomain linker maycomprise SEQ ID NO: 44, corresponding to RAGE amino acids 318-342.

In an embodiment, a fusion protein of the present invention may beadministered by various routes. Administration of the RAGE fusionprotein of the present invention may employ intraperitoneal (IP)injection. Alternatively, the RAGE fusion protein may be administeredorally, intranasally, or as an aerosol. In another embodiment,administration is intravenous (IV). The RAGE fusion protein may also beinjected subcutaneously. In another embodiment, administration of thefusion protein is intra-arterial. In another embodiment, administrationis sublingual. Also, administration may employ a time-release capsule.In yet another embodiment, administration may be transrectal, as by asuppository or the like. For example, subcutaneous administration may beuseful to treat chronic disorders when the self-administration isdesireable.

A variety of animal models have been used to validate the use ofcompounds that modulate RAGE as therapeutics. Examples of these modelsare as follows:

-   -   a) sRAGE inhibited neointimal formation in a rat model of        restenosis following arterial injury in both diabetic and normal        rats by inhibiting endothelial, smooth muscle and macrophage        activation via RAGE (Zhou et al., Circulation 107:2238-2243        (2003));    -   b) Inhibition of RAGE/ligand interactions, using either sRAGE or        an anti-RAGE antibody, reduced amyloid plaque formation in a        mouse model of systemic amyloidosis (Yan et al., Nat. Med.,        6:643-651 (2000)). Accompanying the reduction in amyloid plaques        was a reduction in the inflammatory cytokines, interleukin-6        (IL-6) and macrophage colony stimulating factor (M-CSF) as well        as reduced activation of NF-kB in the treated animals;    -   c) RAGE transgenic mice (RAGE overexpressers and RAGE dominant        negative expressers) exhibit plaque formation and cognitive        deficits in a mouse model of AD (Arancio et al., EMBO J.,        23:4096-4105 (2004));    -   d) Treatment of diabetic rats with sRAGE reduced vascular        permeability (Bonnardel-Phu et al., Diabetes, 48:2052-2058        (1999));    -   e) Treatment with sRAGE reduced atherosclerotic lesions in        diabetic apolipoprotein E-null mice and prevented the functional        and morphological indices of diabetic nephropathy in db/db mice        (Hudson et al., Arch. Biochem. Biophys., 419:80-88 (2003)); and    -   f) sRAGE attenuated the severity of inflammation in a mouse        model of collagen-induced arthritis (Hofmann et al., Genes        Immunol., 3:123-135 (2002)), a mouse model of experimental        allergic encephalomyelitis (Yan et al., Nat. Med. 9:28-293        (2003)) and a mouse model of inflammatory bowel disease (Hofmann        et al., Cell, 97:889-901 (1999)).

Thus, in an embodiment, the fusion proteins of the present invention maybe used to treat a symptom of diabetes and/or complications resultingfrom diabetes mediated by RAGE. In alternate embodiments, the symptom ofdiabetes or diabetic late complications may comprise diabeticnephropathy, diabetic retinopathy, a diabetic foot ulcer, acardiovascular complication of diabetes, or diabetic neuropathy.

Originally identified as a receptor for molecules whose expression isassociated with the pathology of diabetes, RAGE itself is essential tothe pathophysiology of diabetic complications. In vivo, inhibition ofRAGE interaction with its ligand(s) has been shown to be therapeutic inmultiple models of diabetic complications and inflammation (Hudson etal., Arch. Biochem. Biophys., 419:80-88 (2003)). For example, atwo-month treatment with anti-RAGE antibodies normalized kidney functionand reduced abnormal kidney histopathology in diabetic mice (Flyvbjerget al., Diabetes 53:166-172 (2004)). Furthermore, treatment with asoluble form of RAGE (sRAGE) which binds to RAGE ligands and inhibitsRAGE/ligand interactions, reduced atherosclerotic lesions in diabeticapolipoprotein E-null mice and attenuated the functional andmorphological pathology of diabetic nephropathy in db/db mice(Bucciarelli et al., Circulation 106:2827-2835 (2002)).

Also, it has been shown that nonenzymatic glycoxidation ofmacromolecules ultimately resulting in the formation of advancedglycation end products (AGEs) is enhanced at sites of inflammation, inrenal failure, in the presence of hyperglycemia and other conditionsassociated with systemic or local oxidant stress (Dyer et al., J. Clin.Invest., 91:2463-2469 (1993); Reddy et al., Biochem., 34:10872-10878(1995); Dyer et al., J. Biol. Chem., 266:11654-11660 (1991); Degenhardtet al., Cell Mol. Biol., 44:1139-1145 (1998)). Accumulation of AGEs inthe vasculature can occur focally, as in the joint amyloid composed ofAGE-β₂-microglobulin found in patients with dialysis-related amyloidosis(Miyata et al., J. Clin. Invest., 92:1243-1252 (1993); Miyata et al., J.Clin. Invest., 98:1088-1094 (1996)), or generally, as exemplified by thevasculature and tissues of patients with diabetes (Schmidt et al.,Nature Med, 1:1002-1004 (1995)). The progressive accumulation of AGEsover time in patients with diabetes suggests that endogenous clearancemechanisms are not able to function effectively at sites of AGEdeposition. Such accumulated AGEs have the capacity to alter cellularproperties by a number of mechanisms. Although RAGE is expressed at lowlevels in normal tissues and vasculature, in an environment where thereceptor's ligands accumulate, it has been shown that RAGE becomesupregulated (Li et al., J. Biol. Chem., 272:16498-16506 (1997); Li etal., J. Biol. Chem., 273:30870-30878 (1998); Tanaka et al., J. Biol.Chem., 275:25781-25790 (2000)). RAGE expression is increased inendothelium, smooth muscle cells and infiltrating mononuclear phagocytesin diabetic vasculature. Also, studies in cell culture have demonstratedthat AGE-RAGE interaction causes changes in cellular propertiesimportant in vascular homeostasis.

Use of the RAGE fusion proteins in the treatment of diabetes relatedpathology is illustrated in FIG. 13. The RAGE fusion protein TTP-4000was evaluated in a diabetic rat model of restenosis which involvedmeasuring smooth muscle proliferation and intimal expansion followingvascular injury. As illustrated in FIG. 13, TTP-4000 treatment maysignificantly reduce the intima/media (I/M) ratio (FIG. 13A; Table 1) indiabetes-associated restenosis in a dose-responsive manner. Also,TTP-4000 treatment may significantly reduce restenosis-associatedvascular smooth muscle cell proliferation in a dose-responsive manner.TABLE 1 Effect of TTP-4000 in Rat Model of Restenosis TTP-4000 (n = 9)TTP-4000 (n = 9) Low dose** High dose** (0.3 mg/animal (1.0 mg/animalqod IgG (n = 9) qod × 4) qod × 4) Luminal area  0.2 ± 0.03 0.18 ± 0.040.16 ± 0.02 (mm²) Medial area 0.12 ± 0.01 0.11 ± 0.02 0.11 ± 0.01 (mm²)I/M ratio 1.71 ± 0.27 1.61 ± 0.26 1.44* ± 0.15 *P<0.05; **For both high and low dose, a loading dose of 3 mg/animal wasused.

In other embodiments, the fusion proteins of the present invention mayalso be used to treat or reverse amyloidoses and Alzheimer's disease.RAGE is a receptor for amyloid beta (Aβ) as well as other amyloidogenicproteins including SAA and amylin (Yan et al., Nature, 382:685-691(1996); Yan et al., Proc. Natl. Acad. Sci., USA, 94:5296-5301 (1997);Yan et al., Nat. Med., 6:643-651 (2000); Sousa et al., Lab Invest.,80:1101-1110 (2000)). Also, the RAGE ligands, including AGEs, S100b andAβ proteins, are found in tissue surrounding the senile plaque in man(Luth et al., Cereb. Cortex 15:211-220 (2005); Petzold et al, Neurosci.Lett., 336:167-170 (2003); Sasaki et al., Brain Res., 12:256-262 (2001;Yan et al., Restor. Neurol Neruosci., 12:167-173 (1998)). It has beenshown that RAGE binds β-sheet fibrillar material regardless of thecomposition of the subunits (amyloid-β peptide, amylin, serum amyloid A,prion-derived peptide) (Yan et al., Nature, 382:685-691 (1996); Yan etal., Nat. Med., 6:643-651 (2000)). In addition, deposition of amyloidhas been shown to result in enhanced expression of RAGE. For example, inthe brains of patients with Alzheimer's disease (AD), RAGE expressionincreases in neurons and glia (Yan, et al., Nature 382:685-691 (1996)).Concurrent with expression of RAGE ligands, RAGE is upregulated inastrocytes and microglial cells in the hippocampus of individuals withAD but is not upregulated in individuals that do not have AD (Lue etal., Exp. Neurol., 171:29-45 (2001)). These findings suggest that cellsexpressing RAGE are activated via RAGE/RAGE ligand interactions in thevicinity of the senile plaque. Also, in vitro, Aβ-mediated activation ofmicroglial cells can be blocked with antibodies directed against theligand-binding domain of RAGE (Yan et al., Proc. Natl. Acad. Sci., USA,94:5296-5301 (1997)). It has also been demonstrated that RAGE can serveas a focal point for fibril assembly (Deane et al., Nat. Med. 9:907-913(2003)).

Also, in vivo inhibition of RAGE/ligand interactions using either sRAGEor an anti-RAGE antibody can reduce amyloid plaque formation in a mousemodel of systemic amyloidosis (Yan et al., Nat. Med., 6:643-651 (2000)).Double transgenic mice that over-express human RAGE and human amyloidprecursor protein (APP) with the Swedish and London mutations (mutanthAPP) in neurons develop learning defects and neuropathologicalabnormalities earlier than their single mutant hAPP transgeniccounterparts. In contrast, double transgenic mice with diminished Aβsignaling capacity due to neurons expressing a dominant negative form ofRAGE on the same mutant hAPP background, show a delayed onset ofneuropathological and learning abnormalities compared to their singleAPP transgenic counterpart (Arancio et al., EMBO J., 23:4096-4105(2004)).

In addition, inhibition of RAGE-amyloid interaction has been shown todecrease expression of cellular RAGE and cell stress markers (as well asNF-κB activation), and diminish amyloid deposition (Yan et al., Nat.Med., 6:643-651 (2000)) suggesting a role for RAGE-amyloid interactionin both perturbation of cellular properties in an environment enrichedfor amyloid (even at early stages) as well as in amyloid accumulation.

Thus, the RAGE fusion proteins of the present invention may also be usedto treat reduce amyloidosis and to reduce amyloid plaques and cognitivedysfunction associated with Alzheimer's Disease (AD). As describedabove, sRAGE has been shown to reduce both amyloid plaque formation inthe brain and subsequent increase in inflammatory markers in an animalmodel of AD. FIGS. 14A and 14B show that mice that have AD, and aretreated for 3 months with either TTP-4000 or mouse sRAGE had feweramyloid beta (Aβ) plaques and less cognitive dysfunction than animalsthat received a vehicle or a human IgG negative control (IgG1). LikesRAGE, TTP-4000 may also reduce the inflammatory cytokines IL-1 andTNF-α (data not shown) associated with AD.

Also, fusion proteins of the present invention may be used to treatatherosclerosis and other cardiovascular disorders. Thus, it has beenshown that ischemic heart disease is particularly high in patients withdiabetes (Robertson, et al., Lab Invest., 18:538-551 (1968); Kannel etal, J. Am. Med. Assoc., 241:2035-2038 (1979); Kannel et al., Diab. Care,2:120-126 (1979)). In addition, studies have shown that atherosclerosisin patients with diabetes is more accelerated and extensive than inpatients not suffering from diabetes (see e.g. Waller et al., Am. J.Med., 69:498-506 (1980); Crall et al, Am. J. Med. 64:221-230 (1978);Hamby et al., Chest, 2:251-257 (1976); and Pyorala et al., Diab. Metab.Rev., 3:463-524 (1978)). Although the reasons for acceleratedatherosclerosis in the setting of diabetes are many, it has been shownthat reduction of AGEs can reduce plaque formation.

For example, the RAGE fusion proteins of the present invention may alsobe used to treat stroke. When TTP-4000 was compared to sRAGE in adisease relevant animal model of stroke, TTP-4000 was found to provide asignificantly greater reduction in infarct volume. In this model, themiddle carotid artery of a mouse is ligated and then reperfused to forman infart. To assess the efficacy of RAGE fusion proteins to treat orprevent stroke, mice were treated with sRAGE or TTP-4000 or controlimmunoglobulin just prior to reperfusion. As can be seen in Table 2,TTP-4000 was more efficacious than sRAGE in limiting the area of infarctin these animals suggesting that TTP-4000, because of its betterhalf-life in plasma, was able to maintain greater protection than sRAGE.TABLE 2 Reduction of Infarct in Stroke % Reduction of Infarct** sRAGE15%* TTP-4000 (300 μg) 38%* TTP-4000 (300 μg) 21%* TTP-4000 (300 μg)10%* IgG Isotype control  4% (300 μg)*Significant to p<0.001; **Compared to saline

In another embodiment, the fusion proteins of the present invention maybe used to treat cancer. In one embodiment, the cancer treated using thefusion proteins of the present invention comprises cancer cells thatexpress RAGE. For example, cancers that may be treated with the RAGEfusion protein of the present invention include some lung cancers, somegliomas, some papillomas, and the like. Amphoterin is a high mobilitygroup I nonhistone chromosomal DNA binding protein (Rauvala et al., J.Biol. Chem., 262:16625-16635 (1987); Parkikinen et al., J. Biol. Chem.268:19726-19738 (1993)) which has been shown to interact with RAGE. Ithas been shown that amphoterin promotes neurite outgrowth, as well asserving as a surface for assembly of protease complexes in thefibrinolytic system (also known to contribute to cell mobility). Inaddition, a local tumor growth inhibitory effect of blocking RAGE hasbeen observed in a primary tumor model (C6 glioma), the Lewis lungmetastasis model (Taguchi et al., Nature 405:354-360 (2000)), andspontaneously arising papillomas in mice expressing the v-Ha-rastransgene (Leder et al., Proc. Natl. Acad. Sci., 87:9178-9182 (1990)).

In yet another embodiment, fusion proteins of the present invention maybe used to treat inflammation. For example, a\in alternate embodiments,the fusion protein of the present invention is used to treatinflammation associated with autoimmunity, inflammation associated withinflammatory bowel disease, inflammation associated with rheumatoidarthritis, inflammation associated with psoriasis, inflammationassociated with multiple sclerosis, inflammation associated withhypoxia, inflammation associated with stroke, inflammation associatedwith heart attack, inflammation associated with hemorraghic shock,inflammation associated with sepsis, inflammation associated with organtransplantation, or inflammation associated with impaired wound healing.

For example, following thrombolytic treatment, inflammatory cells suchas granulocytes infiltrate the ischemic tissue and produce oxygenradicals that can destroy more cells than were killed by the hypoxia.Inhibiting the receptor on the neutrophil responsible for theneutrophils being able to infiltrate the tissue with antibodies or otherprotein antagonists has been shown to ameliorate the response. SinceRAGE is a ligand for this neutrophil receptor, a fusion proteincontaining a fragment of RAGE may act as a decoy and prevent theneutrophil from trafficking to the reperfused site and thus preventfurther tissue destruction. The role of RAGE in prevention ofinflammation may be indicated by studies showing that sRAGE inhibitedneointimal expansion in a rat model of restenosis following arterialinjury in both diabetic and normal rats, presumably by inhibitingendothelial, smooth muscle cell proliferation and macrophage activationvia RAGE (Zhou et al., Circulation, 107:2238-2243 (2003)). In addition,sRAGE inhibited models of inflammation including delayed-typehypersensitivity, experimental autoimmune encephalitis and inflammatorybowel disease (Hofman et al., Cell, 97:889-901 (1999)).

Also, in an embodiment, the fusion proteins of the present invention maybe used to treat auto-immune based disorders. For example, the fusionproteins of the present invention may be used to treat kidney failure.Thus, the fusion proteins of the present invention may be used to treatsystemic lupus nephritis or inflammatory lupus nephritis. For example,the S100/calgranulins have been shown to comprise a family of closelyrelated calcium-binding polypeptides characterized by two EF-handregions linked by a connecting peptide (Schafer et al., TIBS, 21:134-140(1996); Zimmer et al., Brain Res. Bull., 37:417-429 (1995); Rammes etal., J. Biol. Chem., 272:9496-9502 (1997); Lugering et al., Eur. J.Clin. Invest., 25:659-664 (1995)). Although they lack signal peptides,it has long been known that S100/calgranulins gain access to theextracellular space, especially at sites of chronic immune/inflammatoryresponses, as in cystic fibrosis and rheumatoid arthritis. RAGE is areceptor for many members of the S100/calgranulin family, mediatingtheir proinflammatory effects on cells such as lymphocytes andmononuclear phagocytes. Also, studies on delayed-type hypersensitivityresponse, colitis in IL-10 null mice, collagen-induced arthritis, andexperimental autoimmune encephalitis models suggest that RAGE-ligandinteraction (presumably with S-100/calgranulins) has a proximal role inthe inflammatory cascade.

Thus, in various selected embodiments, the present invention may providea method for inhibiting the interaction of an AGE with RAGE in a subjectby administering to the subject a therapeutically effective amount of afusion protein of the present invention. The subject treated using theRAGE fusion proteins of the present invention may be an animal. In anembodiment, the subject is a human. The subject may be suffering from anAGE-related disease such as diabetes, diabetic complications such asnephropathy, neuropathy, retinopathy, foot ulcer, amyloidoses, or renalfailure, and inflammation. Or, the subject may be an individual withAlzheimer's disease. In an alternative embodiment, the subject may be anindividual with cancer. In yet other embodiments, the subject may besuffering from systemic lupus erythmetosis or inflammatory lupusnephritis. Other diseases may be mediated by RAGE and thus, may betreated using the fusion proteins of the present invention. Thus, inadditional alternative embodiments of the present invention, the fusionproteins may be used for treatment of Crohn's disease, arthritis,vasculitis, nephropathies, retinopathies, and neuropathies in human oranimal subjects.

A therapeutically effective amount may comprise an amount which iscapable of preventing the interaction of RAGE with an AGE or other typesof endogenous RAGE ligands in a subject. Accordingly, the amount willvary with the subject being treated. Administration of the compound maybe hourly, daily, weekly, monthly, yearly, or as a single event. Invarious alternative embodiments, the effective amount of the fusionprotein may range from about 1 ng/kg body weight to about 100 mg/kg bodyweight, or from about 10 μg/kg body weight to about 50 mg/kg bodyweight, or from about 100 μg/kg body weight to about 10 mg/kg bodyweight. The actual effective amount may be established by dose/responseassays using methods standard in the art (Johnson et al., Diabetes. 42:1179, (1993)). Thus, as is known to those in the art, the effectiveamount may depend on bioavailability, bioactivity, and biodegradabilityof the compound.

Compositions

The present invention may comprise a composition comprising a fusionprotein of the present invention mixed with a pharmaceuticallyacceptable carrier. The fusion protein may comprise a RAGE polypeptidelinked to a second, non-RAGE polypeptide. In one embodiment, the fusionprotein may comprise a RAGE ligand binding site. In an embodiment, theligand binding site comprises the most N-terminal domain of the fusionprotein. The RAGE ligand binding site may comprise the V domain of RAGE,or a portion thereof. In an embodiment, the RAGE ligand binding sitecomprises SEQ ID NO: 9 or a sequence 90% identical thereto, or SEQ IDNO: 10 or a sequence 90% identical thereto.

In an embodiment, the RAGE polypeptide may be linked to a polypeptidecomprising an immunoglobulin domain or a portion (e.g., a fragmentthereof) of an immunoglobulin domain. In one embodiment, the thepolypeptide comprising an immunoglobulin domain comprises at least aportion of at least one of the C_(H)2 or the C_(H)3 domains of a humanIgG.

The RAGE protein or polypeptide may comprise full-length human RAGE(e.g., SEQ ID NO: 1), or a fragment of human RAGE. In an embodiment, theRAGE polypeptide does not include any signal sequence residues. Thesignal sequence of RAGE may comprise either residues 1-22 or residues1-23 of full length RAGE (SEQ ID NO: 1). In alternate embodiments, theRAGE polypeptide may comprise a sequence that is 70%, 80% or 90%identical to human RAGE, or a fragment thereof. For example, in oneembodiment, the RAGE polypeptide may comprise human RAGE, or a fragmentthereof, with Glycine as the first residue rather than a Methionine (seee.g., Neeper et al., (1992)). Or, the human RAGE may comprisefull-length RAGE with the signal sequence removed (e.g., SEQ ID NO: 2 orSEQ ID NO: 3) (FIGS. 1A and 1B) or a portion of that amino acidsequence. The fusion proteins of the present invention may also comprisesRAGE (e.g., SEQ ID NO: 4), a polypeptide 90% identical to sRAGE, or afragment of sRAGE. For example, the RAGE polypeptide may comprise humansRAGE, or a fragment thereof, with Glycine as the first residue ratherthan a Methionine (see e.g., Neeper et al., (1992)). Or, the human RAGEmay comprise sRAGE with the signal sequence removed (e.g., SEQ ID NO: 5or SEQ ID NO: 6) (FIG. 1C) or a portion of that amino acid sequence. Inother embodiments, the RAGE protein may comprise a V domain (e.g., SEQID NO: 7 or SEQ ID NO: 8; FIG. 1D). Or, a sequence 90% identical to theV domain or a fragment thereof may be used. Or, the RAGE protein maycomprise a fragment of RAGE comprising a portion of the V domain (e.g.,SEQ ID NO: 9 or SEQ ID NO: 10, FIG. 1D). In an embodiment, the ligandbinding site may comprise SEQ ID NO: 9, or a sequence 90% identicalthereto, or SEQ ID NO: 10, or a sequence 90% identical thereto. In yetanother embodiment, the RAGE fragment is a synthetic peptide.

For example, the RAGE polypeptide may comprise amino acids 23-116 ofhuman RAGE (SEQ ID NO: 7) or a sequence 90% identical thereto, or aminoacids 24-116 of human RAGE (SEQ ID NO: 8) or a sequence 90% identicalthereto, corresponding to the V domain of RAGE. Or, the RAGE polypeptidemay comprise amino acids 124-221 of human RAGE (SEQ ID NO: 11) or asequence 90% identical thereto, corresponding to the C1 domain of RAGE.In another embodiment, the RAGE polypeptide may comprise amino acids227-317 of human RAGE (SEQ ID NO: 12) or a sequence 90% identicalthereto, corresponding to the C2 domain of RAGE. Or, the RAGEpolypeptide may comprise amino acids 23-123 of human RAGE (SEQ ID NO:13) or a sequence 90% identical thereto, or amino acids 24-123 of humanRAGE (SEQ ID NO: 14) or a sequence 90% identical thereto, correspondingto the V domain of RAGE and a downstream interdomain linker. Or, theRAGE polypeptide may comprise amino acids 23-226 of human RAGE (SEQ IDNO: 17) or a sequence 90% identical thereto, or amino acids 24-226 ofhuman RAGE (SEQ ID NO: 18) or a sequence 90% identical thereto,corresponding to the V-domain, the C1 domain and the interdomain linkerlinking these two domains. Or, the RAGE polypeptide may comprise aminoacids 23-339 of human RAGE (SEQ ID NO: 5) or a sequence 90% identicalthereto, or 24-339 of human RAGE (SEQ ID NO: 6) or a sequence 90%identical thereto, corresponding to sRAGE (i.e., encoding the V, C1, andC2 domains and interdomain linkers). Or, fragments of each of thesesequences may be used.

The fusion protein may include several types of peptides that are notderived from RAGE or a fragment thereof. The second polypeptide of thefusion protein may comprise a polypeptide derived from animmunoglobulin. The heavy chain (or portion thereof) may be derived fromany one of the known heavy chain isotypes: IgG (γ), IgM (μ), IgD (δ),IgE (ε), or IgA (α). In addition, the heavy chain (or portion thereof)may be derived from any one of the known heavy chain subtypes: IgG1(γ1), IgG2 (γ2), IgG3 (γ3), IgG4 (γ4), IgA1 (α1), IgA2 (α2), ormutations of these isotypes or subtypes that alter the biologicalactivity. The second polypeptide may comprise the C_(H)2 and C_(H)3domains of a human IgG1 or a portion of either, or both, of thesedomains. As an example embodiments, the polypeptide comprising theC_(H)2 and C_(H)3 domains of a human IgG1 or a portion thereof maycomprise SEQ ID NO: 38 or SEQ ID NO: 40. The immunoglobulin peptide maybe encoded by the nucleic acid sequence of SEQ ID NO: 39 or SEQ ID NO:41.

The Fc portion of the immunoglobulin chain may be proinflammatory invivo. Thus, in one embodiment, the RAGE fusion protein of the presentinvention comprises an interdomain linker derived from RAGE rather thanan interdomain hinge polypeptide derived from an immunoglobulin.

Thus in one embodiment, the fusion protein may further comprise a RAGEpolypeptide directly linked to a polypeptide comprising a C_(H)2 domainof an immunoglobulin, or a fragment thereof. In one embodiment, theC_(H)2 domain, or a fragment thereof comprises SEQ ID NO: 42.

In one embodiment, the RAGE polypeptide comprises a RAGE interdomainlinker linked to a RAGE immunoglobulin domain such that the C-terminalamino acid of the RAGE immunoglobulin domain is linked to the N-terminalamino acid of the interdomain linker, and the C-terminal amino acid ofthe RAGE interdomain linker is directly linked to the N-terminal aminoacid of a polypeptide comprising a C_(H)2 domain of an immunoglobulin,or a fragment thereof. The polypeptide comprising a C_(H)2 domain of animmunoglobulin, or a portion thereof, may comprise the C_(H)2 and C_(H)3domains of a human IgG1. As an example embodiment, the polypeptidecomprising the C_(H)2 and C_(H)3 domains of a human IgG1 may compriseSEQ ID NO: 38 or SEQ ID NO: 40.

The fusion protein of the present invention may comprise a single ormultiple domains from RAGE. Also, the RAGE polypeptide comprising aninterdomain linker linked to a RAGE immunoglobulin domain may comprise afragment of a full-length RAGE protein. For example, in one embodiment,the fusion protein may comprise two immunoglobulin domains derived fromRAGE protein and two immunoglobulin domains derived from a human Fcpolypeptide. The fusion protein may comprise a first RAGE immunoglobulindomain and a first interdomain linker linked to a second RAGEimmunoglobulin domain and a second RAGE interdomain linker, such thatthe N-terminal amino acid of the first interdomain linker is linked tothe C-terminal amino acid of the first RAGE immunoglobulin domain, theN-terminal amino acid of the second RAGE immunoglobulin domain is linkedto C-terminal amino acid of the first interdomain linker, the N-terminalamino acid of the second interdomain linker is linked to C-terminalamino acid of the RAGE second immunoglobulin domain, and the C-terminalamino acid of the RAGE second interdomain linker is directly linked tothe N-terminal amino acid of the polypeptide comprising a C_(H)2immunoglobulin domain or fragment thereof. For example, the RAGEpolypeptide may comprise amino acids 23-251 of human RAGE (SEQ ID NO:19) or a sequence 90% identical thereto, or amino acids 24-251 of humanRAGE (SEQ ID NO: 20) or a sequence 90% identical thereto, correspondingto the V-domain, the C1 domain, the interdomain linker linking these twodomains, and a second interdomain linker downstream of C1. In oneembodiment, a nucleic acid construct comprising SEQ ID NO: 30 or afragment thereof may encode for a four domain RAGE fusion protein.

Alternatively, a three domain fusion protein may comprise oneimmunoglobulin domain derived from RAGE and two immunoglobulin domainsderived from a human Fc polypeptide. For example, the fusion protein maycomprise a single RAGE immunoglobulin domain linked via a RAGEinterdomain linker to the N-terminal amino acid of the polypeptidecomprising a C_(H)2 immunoglobulin domain or a fragment thereof. Forexample, the RAGE polypeptide may comprise amino acids 23-136 of humanRAGE (SEQ ID NO: 15) or a sequence 90% identical thereto or amino acids24-136 of human RAGE (SEQ ID NO: 16) or a sequence 90% identical theretocorresponding to the V domain of RAGE and a downstream interdomainlinker. In one embodiment, a nucleic acid construct comprising SEQ IDNO: 31 or a fragment thereof may encode for a three domain RAGE fusionprotein.

A RAGE interdomain linker fragment may comprise a peptide sequence thatis naturally downstream of, and thus, linked to, a RAGE immunoglobulindomain. For example, for the RAGE V domain, the interdomain linker maycomprise amino acid sequences that are naturally downstream from the Vdomain. In an embodiment, the linker may comprise SEQ ID NO: 21,corresponding to amino acids 117-123 of full-length RAGE. Or, the linkermay comprise a peptide having additional portions of the natural RAGEsequence. For example, a interdomain linker comprising several aminoacids (e.g., 1-3, 1-5, or 1-10, or 1-15 amino acids) upstream anddownstream of SEQ ID NO: 21 may be used. Thus, in one embodiment, theinterdomain linker comprises SEQ ID NO: 23 comprising amino acids117-136 of full-length RAGE. Or, fragments of SEQ ID NO: 21 deleting,for example, 1, 2, or 3, amino acids from either end of the linker maybe used. In alternate embodiments, the linker may comprise a sequencethat is 70% identical, or 80% identical, or 90% identical to SEQ ID NO:21 or SEQ ID NO: 23.

For the RAGE C1 domain, the linker may comprise peptide sequence that isnaturally downstream of the C1 domain. In an embodiment, the linker maycomprise SEQ ID NO: 22, corresponding to amino acids 222-251 offull-length RAGE. Or, the linker may comprise a peptide havingadditional portions of the natural RAGE sequence. For example, a linkercomprising several (1-3, 1-5, or 1-10, or 1-15 amino acids) amino acidsupstream and downstream of SEQ ID NO: 22 may be used. Or, fragments ofSEQ ID NO: 22 may be used, deleting for example, 1-3, 1-5, or 1-10, or1-15 amino acids from either end of the linker. For example, in oneembodiment, a RAGE interdomain linker may comprise SEQ ID NO: 24,corresponding to amino acids 222-226. Or an interdomain linker maycomprise SEQ ID NO: 44, corresponding to RAGE amino acids 318-342.

Pharmaceutically acceptable carriers may comprise any of the standardpharmaceutically accepted carriers known in the art. The carrier maycomprise a diluent. In one embodiment, the pharmaceutical carrier may bea liquid and the fusion protein or nucleic acid construct may be in theform of a solution. In another embodiment, the pharmaceuticallyacceptable carrier may be a solid in the form of a powder, a lyophilizedpowder, or a tablet. Or, the pharmaceutical carrier may be a gel,suppository, or cream. In alternate embodiments, the carrier maycomprise a liposome, a microcapsule, a polymer encapsulated cell, or avirus. Thus, the term pharmaceutically acceptable carrier encompasses,but is not limited to, any of the standard pharmaceutically acceptedcarriers, such as water, alcohols, phosphate buffered saline solution,sugars (e.g., sucrose or mannitol), oils or emulsions such as oil/wateremulsions or a trigyceride emulsion, various types of wetting agents,tablets, coated tablets and capsules.

Administration of the RAGE fusion proteins of the present invention mayemploy various routes. Thus, administration of the RAGE fusion proteinof the present invention may employ intraperitoneal (IP) injection.Alternatively, the RAGE fusion protein may be administered orally,intranasally, or as an aerosol. In another embodiment, administration isintravenous (IV). The RAGE fusion protein may also be injectedsubcutaneously. In another embodiment, administration of the fusionprotein is intra-arterial. In another embodiment, administration issublingual. Also, administration may employ a time-release capsule. Inyet another embodiment, administration may be transrectal, as by asuppository or the like. For example, subcutaneous administration may beuseful to treat chronic disorders when the self-administration isdesireable.

The pharmaceutical compositions may be in the form of a sterileinjectable solution in a non-toxic parenterally acceptable solvent orvehicle. Among the acceptable vehicles and solvents that may be employedare water, Ringer's solution, 3-butanediol, isotonic sodium chloridesolution, or aqueous buffers, as for example, physiologically acceptablecitrate, acetate, glycine, histidine, phosphate, tris or succinatebuffers. The injectable solution may contain stabilizers to protectagainst chemical degradation and aggregate formation. Stabilizers mayinclude antioxidants such as butylated hydroxy anisole (BHA), andbutylated hydroxy toluene (BHT), buffers (citrates, glycine, histidine)or surfactants (polysorbate 80, poloxamers). The solution may alsocontain antimicrobial preservatives, such as benzyl alcohol andparabens. The solution may also contain surfactants to reduceaggregation, such as Polysorbate 80, poloxomer, or other surfactantsknown in the art. The solution may also contain other additives, such asa sugar(s) or saline, to adjust the osmotic pressure of the compositionto be similar to human blood.

The pharmaceutical compositions may be in the form of a sterilelyophilized powder for injection upon reconstitution with a diluent. Thediluent can be water for injection, bacteriostatic water for injection,or sterile saline. The lyophilized powder may be produced by freezedrying a solution of the fusion protein to produce the protein in dryform. As is known in the art, the lyophilized protein generally hasincreased stability and a longer shelf life than a liquid solution ofthe protein. The lyophilized powder (cake) many contain a buffer toadjust the pH, as for example physiologically acceptable citrate,acetate, glycine, histidine, phosphate, tris or succinate buffer. Thelyophilized powder may also contain lyoprotectants to maintain itsphysical and chemical stability. The commonly used lyoprotectants arenon-reducing sugars and disaccharides such as sucrose, mannitol, ortrehalose. The lyophilized powder may contain stabilizers to protectagainst chemical degradation and aggregate formation. Stabilizers mayinclude, but are not limited to antioxidants (BHA, BHT), buffers(citrates, glycine, histidine), or surfactants (polysorbate 80,poloxamers). The lyophilized powder may also contain antimicrobialpreservatives, such as benzyl alcohol and parabens. The lyophilizedpowder may also contain surfactants to reduce aggregation, such as, butnot limited to, Polysorbate 80 and poloxomer. The lyophilized powder mayalso contain additives (e.g., sugars or saline) to adjust the osmoticpressure to be similar to human blood upon reconstitution of the powder.The lyophilized powder may also contain bulking agents, such as sugarsand disaccharides.

The pharmaceutical compositions for injection may also be in the form ofa oleaginous suspension. This suspension may be formulated according tothe known methods using suitable dispersing or wetting agents andsuspending agents described above. In addition, sterile, fixed oils areconveniently employed as solvent or suspending medium. For this purpose,any bland fixed oil may be employed using synthetic mono- ordiglycerides. Also, oily suspensions may be formulated by suspending theactive ingredient in a vegetable oil, for example arachis oil, oliveoil, sesame oil or coconut oil, or in a mineral oil such as a liquidparaffin. For example, fatty acids such as oleic acid find use in thepreparation of injectables. The oily suspensions may contain athickening agent, for example beeswax, hard paraffin or cetyl alchol.These compositions may be preserved by the addition of an anti-oxidantsuch as ascorbic acid.

The pharmaceutical compositions of the present invention may also be inthe form of oil-in-water emulsions or aqueous suspensions. The oilyphase may be a vegetable oil, for example, olive oil or arachis oil, ora mineral oil, for example a liquid paraffin, or a mixture thereof.Suitable emulsifying agents may be naturally-occurring gums, for examplegum acacia or gum tragacanth, naturally-occurring phosphatides, forexample soy bean, lecithin, and esters or partial esters derived fromfatty acids and hexitol anhydrides, for example sorbitan monooleate, andcondensation products of said partial esters with ethylene oxide, forexample polyoxyethylene sorbitan.

Aqueous suspensions may also contain the active compounds in admixturewith excipients. Such excipients may include suspending agents, forexample sodium carboxymethylcellulose, methylcellulose,hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gumtragacanth and gum acacia; dispersing or wetting agents, such as anaturally-occurring phosphatide such as lecithin, or condensationproducts of an alkylene oxide with fatty acids, for examplepolyoxyethylene stearate, or condensation products of ethylene oxidewith long chain aliphatic alcohols, for example,heptadecaethyl-eneoxycetanol, or condensation products of ethylene oxidewith partial esters derived from fatty acids and a hexitol such aspolyoxyethylene sorbitol monooleate, or condensation products ofethylene oxide with partial esters derived from fatty acids and hexitolanhydrides, for example polyethylene sorbitan monooleate.

Dispersible powders and granules suitable for preparation of an aqueoussuspension by the addition of water may provide the active compound inadmixture with a dispersing agent, suspending agent, and one or morepreservatives. Suitable preservatives, dispersing agents, and suspendingagents are described above.

The compositions may also be in the form of suppositories for rectaladministration of the compounds of the invention. These compositions canbe prepared by mixing the drug with a suitable non-irritating excipientwhich is solid at ordinary temperatures but liquid at the rectaltemperature and will thus melt in the rectum to release the drug. Suchmaterials include cocoa butter and polyethylene glycols, for example.

For topical use, creams, ointments, jellies, solutions or suspensionscontaining the compounds of the invention may be used. Topicalapplications may also include mouth washes and gargles. Suitablepreservatives, antioxidants such as BHA and BHT, dispersants,surfactants, or buffers may be used.

The compounds of the present invention may also be administered in theform of liposome delivery systems, such as small unilamellar vesicles,large unilamellar vesicles, and multilamellar vesicles. Liposomes may beformed from a variety of phospholipids, such as cholesterol,stearylamine, or phosphatidylcholines.

In certain embodiments, the compounds of the present invention may bemodified to further retard clearance from the circulation by metabolicenzymes. In one embodiment, the compounds may be modified by thecovalent attachment of water-soluble polymers such as polyethyleneglycol (PEG), copolymers of PEG and polypropylene glycol,polyvinylpyrrolidone or polyproline, carboxymethyl cellulose, dextran,polyvinyl alcohol, and the like. Such modifications also may increasethe compound's solubility in aqueous solution. Polymers such as PEG maybe covalently attached to one or more reactive amino residues, sulfydrylresidues or carboxyl residues. Numerous activated forms of PEG have beendescribed, including active esters of carboxylic acid or carbonatederivatives, particularly those in which the leaving groups areN-hydroxsuccinimide, p-nitrophenol, imdazole or1-hydroxy-2-nitrobenzene-3 sulfone for reaction with amino groups,multimode or halo acetyl derivatives for reaction with sulfhydrylgroups, and amino hydrazine or hydrazide derivatives for reaction withcarbohydrate groups.

Additional methods for preparation of protein formulations which may beused with the fusion proteins of the present invention are described inU.S. Pat. Nos. 6,267,958, and 5,567,677.

In a further aspect of the present invention, the RAGE modulators of theinvention are utilized in adjuvant therapeutic or combinationtherapeutic treatments with other known therapeutic agents. Thefollowing is a non-exhaustive listing of adjuvants and additionaltherapeutic agents which may be utilized in combination with the RAGEfusion protein modulators of the present invention:

Pharmacologic Classifications of Anticancer Agents:

-   1. Alkylating agents: Cyclophosphamide, nitrosoureas, carboplatin,    cisplatin, procarbazine-   2. Antibiotics: Bleomycin, Daunorubicin, Doxorubicin-   3. Antimetabolites: Methotrexate, Cytarabine, Fluorouracil-   4. Plant alkaloids: Vinblastine, Vincristine, Etoposide, Paclitaxel,-   5. Hormones: Tamoxifen, Octreotide acetate, Finasteride, Flutamide-   6. Biologic response modifiers: Interferons, Interleukins,

Pharmacologic Classifications of Rreatment for Rheumatoid Arthritis

-   1. Analgesics: Aspirin-   2. NSAIDs (Nonsteroidal anti-inflammatory drugs): Ibuprofen,    Naproxen, Diclofenac-   3. DMARDs (Disease-Modifying Antirheumatic drugs): Methotrexate,    gold preparations, hydroxychloroquine, sulfasalazine-   4. Biologic Response Modifiers, DMARDs: Etanercept, Infliximab    Glucocorticoids

Pharmacologic Classifications of Treatment for Diabetes Mellitus

-   1. Sulfonylureas: Tolbutamide, Tolazamide, Glyburide, Glipizide-   2. Biguanides: Metformin-   3. Miscellaneous oral agents: Acarbose, Troglitazone-   4. Insulin

Pharmacologic Classifications of Treatment for Alzheimer's Disease

-   1. Cholinesterase Inhibitor: Tacrine, Donepezil-   2. Antipsychotics: Haloperidol, Thioridazine-   3. Antidepressants: Desipramine, Fluoxetine, Trazodone, Paroxetine-   4. Anticonvulsants: Carbamazepine, Valproic acid

In one embodiment, the present invention may therefore provide a methodof treating RAGE mediated diseases, the method comprising administeringto a subject in need thereof, a therapeutically effective amount of aRAGE fusion protein in combination with therapeutic agents selected fromthe group consisting of alkylating agents, antimetabolites, plantalkaloids, antibiotics, hormones, biologic response modifiers,analgesics, NSAIDs, DMARDs, glucocorticoids, sulfonylureas, biguanides,insulin, cholinesterase inhibitors, antipsychotics, antidepressants, andanticonvulsants. In a further embodiment, the present invention providesthe pharmaceutical composition of the invention as described above,further comprising one or more therapeutic agents selected from thegroup consisting of alkylating agents, antimetabolites, plant alkaloids,antibiotics, hormones, biologic response modifiers, analgesics, NSAIDs,DMARDs, glucocorticoids, sulfonylureas, biguanides, insulin,cholinesterase inhibitors, antipsychotics, antidepressants, andanticonvulsants.

EXAMPLES

Features and advantages of the inventive concept covered by the presentinvention are further illustrated in the examples which follow.

Example 1 Production of RAGE-IgG Fc Fusion Proteins

Two plasmids were constructed to express RAGE-IgG Fc fusion proteins.Both plasmids were constructed by ligating different lengths of a 5′cDNA sequence from human RAGE with the same 3′ cDNA sequence from humanIgG Fc (γ1). These expression sequences (i.e., ligation products) werethen inserted in pcDNA3.1 expression vector (Invitrogen, Calif.). Thenucleic acid sequences that encode the fusion protein coding region areshown in FIGS. 2 and 3. For TTP-4000 fusion protein, the nucleic acidsequence from 1 to 753 (highlighted in bold) encodes the RAGE N-terminalprotein sequence, whereas the nucleic acid sequence from 754 to 1386encodes the IgG Fc protein sequence (FIG. 2). For TTP-3000, the nucleicacid sequence from 1 to 408 (highlighted in bold) encodes the RAGEN-terminal protein sequence, whereas the nucleic acid sequence from 409to 1041 encodes the IgG Fc protein sequence (FIG. 3).

To produce the RAGE fusion proteins, the expression vectors comprisingthe nucleic acid sequences of either SEQ ID NO: 30 or SEQ ID NO: 31 werestably transfected into CHO cells. Positive transformants were selectedfor neomycin resistance conferred by the plasmid and cloned. Highproducing clones as detected by Western Blot analysis of supernatantwere expanded and the gene product was purified by affinitychromatography using Protein A columns. Expression was optimized so thatcells were producing recombinant TTP-4000 at levels of about 1.3 gramsper liter.

The expressed polypeptides encoding the two fusion proteins areillustrated in FIGS. 4-6. For the four domain structure of TTP-4000, thefirst 251 amino acids (shown in bold in FIG. 4) contain a signalsequence (1-22/23), the V immunoglobulin (and ligand binding) domain(23/24-116), a second interdomain linker (117-123), a secondimmunoglobulin domain (C_(H)1) (124-221), and a second linker (222-251)of the human RAGE protein (FIGS. 4, 6B). The sequence from 252 to 461includes the C_(H)2 and C_(H)3 immunoglobulin domains of IgG.

For the three domain structure of TTP-3000, the first 136 amino acids(shown in bold) contain a signal sequence (1-22/23), the Vimmunoglobulin (and ligand binding) domain (23/24-116) and aninterdomain linker sequence (117-136) of the human RAGE protein (FIGS.5, 6B). In addition, for TT3, the sequence from 137 to 346 includes theC_(H)2 and C_(H)3 immunoglobulin domains of IgG.

Example 2 Method for Testing Activity of a RAGE-IgG1 Fusion Protein

A. In Vitro Ligand Binding:

Known RAGE ligands were coated onto the surface of Maxisorb plates at aconcentration of 5 micrograms per well. Plates were incubated at 4° C.overnight. Following ligand incubation, plates were aspirated and ablocking buffer of 1% BSA in 50 mM imidizole buffer (pH 7.2) was addedto the plates for 1 hour at room temperature. The plates were thenaspirated and/or washed with wash buffer (20 mM Imidizole, 150 mM NaCl,0.05% Tween-20, 5 mM CaCl₂ and 5 mM MgCl₂, pH 7.2). A solution ofTTP-3000 (TT3) at an initial concentration of 1.082 mg/mL and a solutionof TTP-4000 (TT4) at an initial concentration of 370 μg/mL wereprepared. The fusion protein was added at increasing dilutions of theinitial sample. The RAGE fusion protein was allowed to incubate with theimmobilized ligand at 37° C. for one hour after which the plate waswashed and assayed for binding of the fusion protein. Binding wasdetected by the addition of an immunodetection complex containing amonoclonal mouse anti-human IgG1 diluted 1:11,000 to a final assayconcentration (FAC) of 21 ng/100 μL, a biotinylated goat anti-mouse IgGdiluted 1:500, to a FAC of 500 ng/μL, and an avidin-linked alkalinephosphatase. The complex was incubated with the immobilized fusionprotein for one hour at room temperature after which the plate waswashed and the alkaline phosphatase substrate para-nitrophenylphosphate(PNPP) was added. Binding of the complex to the immobilized fusionprotein was quantified by measuring conversion of PNPP topara-nitrophenol (PNP) which was measured spectrophotometrically at 405nm.

As illustrated in FIG. 7, the fusion proteins TTP-4000 (TT4) andTTP-3000 (TT3) specifically interact with known RAGE ligandsamyloid-beta (Abeta), S100b (S100), and amphoterin (Ampho). In theabsence of ligand, i.e., BSA coating alone (BSA or BSA+wash) there wasno increase in absorbance over levels attributable to non-specificbinding of the immunodetection complex. Where amyloid beta is used asthe labeled ligand it may be necessary to preincubate the amyloid betabefore the assay. Preincubation may allow the amyloid beta toself-aggregate into pleated sheet form, as amyloid beta maypreferentially bind to RAGE in the form of a pleated sheet.

Additional evidence for a specific interaction between RAGE fusionproteins TTP-4000 and TTP-3000 with RAGE ligands is exemplified instudies showing that a RAGE ligand is able to effectively compete with aknown RAGE ligand for binding to the fusion proteins. In these studies,amyloid-beta (A-beta) was immobilized on a Maxisorb plate and fusionprotein added as described above. In addition, a RAGE ligand was addedto some of the wells at the same time as the fusion protein.

It was found that the RAGE ligand could block binding of TTP-4000 (TT4)by about 25% to 30% where TTP-4000 was present at 123 μg/mL (1:3dilution, FIG. 8). When the initial solution of TTP-4000 was diluted bya factor of 10 or 30 (1:10 or 1:30), binding of the fusion protein tothe immobilized ligand was completely inhibited by the RAGE ligand.Similarly, the RAGE ligand blocked binding of TTP-3000 (TT3) by about50% where TTP-3000 was present at 360 μg/mL (1:3 dilution, FIG. 9). Whenthe initial solution of TTP-3000 was diluted by a factor of 10 (1:10),binding of the fusion protein to the immobilized ligand was completelyinhibited by the RAGE ligand. Thus, specificity of binding of the RAGEfusion protein to the RAGE ligand was dose dependent. Also, as shown inFIGS. 8 and 9, there was essentially no binding detected in the absenceof fusion protein, i.e., using only the immunodetection complex(“Complex alone”).

B. Effect of RAGE Fusion Proteins in a Cell Based Assay

Previous work has shown that the myeloid THP-1 cells may secrete TNF-αin response to RAGE ligands. In this assay, THP-1 cells were cultured inRPMI-1640 media supplemented with 10% FBS using a protocol provided byATCC. The cells were induced to secrete TNF-α via stimulation of RAGEwith 0.1 mg/ml S100b both in the absence and the presence of the fusionproteins TTP-3000 (TT3) or TTP-4000 (TT4) (10 μg), sRAGE (10 μg), and ahuman IgG (10 μg) (i.e., as a negative control). The amount of TNF-αsecreted by the THP-1 cells was measured 24 hours after the addition ofthe proteins to the cell culture using a commercially available ELISAkit for TNF-α (R&D Systems, Minneapolis, Minn.). The results in FIG. 10demonstrate that the fusion proteins inhibit the S100b/RAGE-inducedproduction of TNF-α in these cells. As shown in FIG. 10, upon additionof 10 μg TTP-3000 or TTP-4000 RAGE fusion protein, induction of TNF-α byS100b (0.1 mg/ml FAC) was reduced by about 45% to 70%, respectively.Fusion protein TTP-4000 may be at least as effective in blocking S100binduction of TNF-α as is sRAGE (FIG. 10). Specificity of the inhibitionfor the RAGE sequences of TTP-4000 and TTP-3000 is shown by theexperiment in which IgG alone was added to S100b stimulated cells.Addition of IgG and S100b to the assay shows the same levels of TNF-α asS100b alone. Specificity of the inhibition of TNF-α induction byTTP-4000 and TTP-3000 for RAGE sequences of the fusion protein is shownby an experiment in which IgG alone was added to S100b stimulated cells.It can be seen that the addition of IgG, i.e., human IgG without theRAGE sequence (Sigma human IgG added at 10 μg/well), and S100b to theassay shows the same levels of TNF-α as S100b alone.

Example 3 Pharmacokinetic Profile of TTP-4000

To determine whether TTP-4000 would have a superior pharmacokineticprofile as compared to human sRAGE, rats and nonhuman primates weregiven an intravenous (IV) injection of TTP-4000 (5 mg/kg) and thenplasma was assessed for the presence of TTP-4000. In these experiments,two naïve male monkeys received a single IV bolus dose of TTP-4000 (5mg/ml/kg) in a peripheral vein followed by an approximate 1.0 milliliter(mL) saline flush. Blood samples (approximately 1.0 mL) were collectedat pre-dose (i.e., prior to injection of the TTP-4000), or at 0.083,0.25, 0.5, 2, 4, 8, 12, 24, 48, 72, 96, 120, 168, 240, 288, and 336hours post dose into tubes containing (lithium heparin). Followingcollection, the tubes were placed on wet ice (maximum 30 minutes) untilcentrifugation under refrigeration (at 2 to 8° C.) at 1500×g for 15minutes. Each harvested plasma sample was then stored frozen (−70°C.±10° C.) until assayed for RAGE polypeptide using an ELISA at varioustime-points following the injection, as described in Example 6.

The kinetic profile shown in FIG. 11 reveals that once TTP-4000 hassaturated its ligands as evidenced by the fairly steep slope of thealpha phase in 2 animals, it retains a terminal half-life of greaterthan 300 hours. This half-life is significantly greater than thehalf-life of human sRAGE in plasma (generally about 2 hours) andprovides an opportunity for single injections for acute and semi-chronicindications. In FIG. 11 each curve represents a different animal underthe same experimental conditions.

Example 4 TTP-4000 Fc Activation

Experiments were performed to measure the activation of the Fc receptorby RAGE fusion protein TTP-4000 as compared to human IgG. Fc receptoractivation was measured by measuring TNF-α secretion from THP-1 cellsthat express the Fc receptor. In these experiments, a 96 well plate wascoated with 10 μg/well TTP-4000 or human IgG. Fc stimulation results inTNF-α secretion. The amount of TNF-α was measured by an Enzyme LinkedImmunoabsorbent Assay (ELISA).

Thus, in this assay, the myeloid cell line, THP-1 (ATTC # TIB-202) wasmaintained in RPMI-1640 media supplemented with 10% fetal bovine serumper ATCC instructions. Typically, 40,000-80,000 cells per well wereinduced to secrete TNF-alpha via Fc receptor stimulation by precoatingthe well with 10 ug/well of either heat aggregated (63° C. for 30 min)TTP-4000 or human IgG1. The amount of TNF-alpha secreted by the THP-1cells was measured in supernatants collected from 24 hours cultures ofcells in the treated wells using a commercially available TNF ELISA kit(R&D Systems, Minneapolis, Minn. # DTA00C) per instructions.

Results are shown in FIG. 12 where it can be seen that TTP-4000generates less than 2 ng/well TNF and IgG generated greater than 40ng/well.

Example 5 In Vivo Activity of TTP-4000

The activity of TTP-4000 was compared to sRAGE in several in vivo modelsof human disease.

A. TTP-4000 in an Animal Model of Restenosis

The RAGE fusion protein TTP-4000 was evaluated in a diabetic rat modelof restenosis which involved measuring smooth muscle proliferation andintimal expansion 21 days following vascular injury. In theseexperiments, balloon injury of left common carotid artery was performedin Zucker diabetic and nondiabetic rats using standard procedure. Aloading dose (3 mg/rat) of IgG, TTP-4000 or phosphate buffered saline(PBS) was administered intraperitoneally (IP) one day prior injury. Amaintenance dose was delivered every other day until day 7 after injury(i.e., at day 1, 3, 5 and 7 after injury). The maintenance dose washigh=1 mg/animal for one group, or low=0.3 mg/animal for the secondgroup. To measure vascular smooth muscle cell (VSMC) proliferation,animals were sacrificed at 4 days and 21 days after injury.

For the measurement of cell proliferation, 4 day animals receivedintraperitoneal injection of bromodeoxyuridine (BrDdU) 50 mg/kg at 18,12, and 2 hours before euthanasia. After sacrifice, the entire left andright carotid arteries were harvested. Specimens were stored inHistochoice for at least 24 hours before embedding. Assessment of VSMCproliferation was performed using mouse anti-BrdU monoclonal antibody. Afluorescence labeled goat anti-mouse secondary antibody was applied. Thenumber of BrdU-positive nuclei per section were counted by two observersblinded to the treatment regimens.

The remaining rats were sacrificed at 21 days for morphometric analysis.Morphometric analyses were performed by an observer blinded to the studygroups, using computerized digital microscopic planimetry softwareImage-Pro Plus on serial sections, (5 mm apart) carotid arteries stainedby Van Gieson staining. All data were expressed as mean±SD. Statisticalanalysis was performed with use of SPSS software. Continuous variableswere compared using unpaired t tests. A values of P≦0.05 was consideredto be statistically significant.

As seen in FIGS. 13A and 13B, TTP-4000 treatment significantly reducedthe intima/media ratio and vascular smooth muscle cell proliferation ina dose-responsive fashion. In FIG. 13 B, the y-axis represents thenumber of BrdU proliferating cells.

B. TTP4000 in an Animal Model of AD

Experiments were performed to evaluate whether TTP-4000 could affectamyloid formation and cognitive dysfunction in a mouse model of AD. Theexperiments utilized transgenic mice expressing the human Swedish mutantamyloid precursor protein (APP) under the control of the PDGF-B chainpromoter. Over time, these mice generate high levels of the RAGE ligand,amyloid beta (Aβ). Previously, sRAGE treatment for 3 months has beenshown to reduce both amyloid plaque formation in the brain and theassociated increase in inflammatory markers in this model.

The APP mice (male) used in this experiment were designed bymicroinjection of the human APP gene (with the Swedish and Londonmutations) into mouse eggs under the control of the platelet-derivedgrowth factor B (PDGF-B) chain gene promoter. The mice were generated ona C57BL/6 background and were developed by Molecular Therapeutics Inc.Animals were fed ad libitum and maintained by brother sister mating. Themice generated from this construct develop amyloid deposits starting at6 months of age. Animals were aged for 6 months and then maintained for90 days and sacrificed for amyloid quantification.

APP transgenic mice were administered vehicle or TTP4000 every other day[qod (i.p.)] for 90 days starting at 6 months of age. At the end of theexperiment, animals were sacrificed and examined for AD plaque burden inthe brain (i.e., plaque number). A 6-month control APP group was used todetermine the baseline of amyloid deposits. In addition, at the end ofthe study, the animals were subjected to behavioral (Morris water maze)analysis. The investigators were blinded to the study compounds. Sampleswere given to the mice at 0.25 ml/mouse/every other day. In addition,one group of mice were given 200 ug/day of human sRAGE.

1. Amyloid Beta Deposition

For histological examination, the animals were anesthetized with anintraperitoneal injection (IP) of sodium pentobarbital (50 mg/kg). Theanimals were transcardially perfused with 4° C., phosphate-bufferedsaline (PBS) followed by 4% paraformaldehyde. The brains were removedand placed in 4% paraformaldehyde over night. The brains were processedto paraffin and embedded. Ten serial 30-μm thick sections through thebrain were obtained. Sections were subjected to primary antibodyovernight at 4° C. (Aβ peptide antibody) in order to detect the amyloiddeposits in the brain of the transgenic animals (Guo et al., J.Neurosci., 22:5900-5909 (2002)). Sections were washed in Tris-bufferedsaline (TBS) and secondary antibody was added and incubated for 1 hourat room temperature. After washing, the sections were incubated asinstructed in the Vector ABC Elite kit (Vector Laboratories) and stainedwith diaminobenzoic acid (DAB). The reactions were stopped in water andcover-slipped after treatment with xylene. The amyloid area in eachsection was determined with a computer-assisted image analysis system,consisting of a Power Macintosh computer equipped with a Quick Captureframe grabber card, Hitachi CCD camera mounted on an Olympus microscopeand camera stand. NIH Image Analysis Software, v. 1.55 was used. Theimages were captured and the total area of amyloid was determined overthe ten sections. A single operator blinded to treatment statusperformed all measurements. Summing the amyloid volumes of the sectionsand dividing by the total number of sections was done to calculate theamyloid volume.

For quantitative analysis, an enzyme-linked immunosorbent assay (ELISA)was used to measure the levels of human total Aβ, Aβ_(total) and Aβ₁₋₄₂in the brains of APP transgenic mice (Biosource International,Camarillo, Calif.). Aβ_(total) and Aβ₁₋₄₂ were extracted from mousebrains by guanidine hydrochloride and quantified as described by themanufacturer. This assay extracts the total Aβ peptide from the brain(both soluble and aggregated).

2. Cognitive Function

The Morris water-maze testing was performed as follows: All mice weretested once in the Morris water maze test at the end of the experiment.Mice were trained in a 1.2 m open field water maze. The pool was filledto a depth of 30 cm with water and maintained at 25° C. The escapeplatform (10 cm square) was placed 1 cm below the surface of the water.During the trials, the platform was removed from the pool. The cued testwas carried out in the pool surrounded with white curtains to hide anyextra-maze cues. All animals underwent non-spatial pretraining (NSP) forthree consecutive days. These trials are to prepare the animals for thefinal behavioral test to determine the retention of memory to find theplatform. These trials were not recorded, but were for training purposesonly. For the training and learning studies, the curtains were removedto extra maze cues (this allowed for identification of animals withswimming impairments). On day 1, the mice were placed on the hiddenplatform for 20 seconds (trial 1), for trials 2-3 animals were releasedin the water at a distance of 10 cm from the cued-platform or hiddenplatform (trial 4) and allowed to swim to the platform. On the secondday of trails, the hidden platform was moved randomly between the centerof the pool or the center of each quadrant. The animals were releasedinto the pool, randomly facing the wall and were allowed 60 seconds toreach the platform (3 trials). In the third trial, animals were giventhree trials, two with a hidden platform and one with a cued platform.Two days following the NSP, animals were subjected to final behavioraltrials (Morris water maze test). For these trials (3 per animal), theplatform was placed in the center of one quadrant of the pool and theanimals released facing the wall in a random fashion. The animal wasallowed to find the platform or swim for 60 seconds (latency period, thetime it takes to find the platform). All animals were tested within 4-6hours of dosing and were randomly selected for testing by an operatorblinded to the test group.

The results are expressed as the mean±standard deviations (SD). Thesignificance of differences in the amyloid and behavioral studies wereanalyzed using a t-test. Comparisons were made between the 6-month-oldAPP control group and the TTP-4000 treated animals, as well as, the9-month-old APP vehicle treated group and the TTP-4000 treated animals.Differences below 0.05 were considered significant. Percent changes inamyloid and behavior were determined by taking the summation of the datain each group and dividing by the comparison (i.e., 1, i.p./6 monthcontrol=% change).

FIGS. 14A and 14B show that mice treated for 3 months with eitherTTP-4000 or mouse sRAGE had fewer Aβ plaques and less cognitivedysfunction than vehicle and negative control human IgG1 (IgG1) treatedanimals. This data indicates that TTP-4000 is effective in reducing ADpathology in a transgenic mouse model. It was also found that likesRAGE, TTP-4000 can reduce the inflammatory cytokines IL-1 and TNF-α(data not shown).

C. Efficacy of TTP-4000 in an Animal Model of Stroke

TTP-4000 was also compared to sRAGE in a disease relevant animal modelof stroke. In this model, the middle carotid artery of a mouse wasligated for 1 hour followed by 23 hours of reperfusion at which pointthe mice were sacrificed and the area of the infarct in the brain wasassessed. Mice were treated with sRAGE or TTP-4000 or controlimmunoglobulin just prior to reperfusion.

In these experiments, male C57BL/6 were injected with vehicle at 250μl/mouse or TTP test articles (TTP-3000, TTP-4000 at 250 μl/mouse). Micewere injected intraperitoneally, 1 hour after the initiation ofischemia. Mice were subjected to one hour of cerebral ischemia followedby 24 hours of reperfusion. To induce ischemia, each mouse wasanesthetized and body temperature was maintained at 36-37° C. byexternal warming. The left common carotid artery (CCA) was exposedthrough a midline incision in the neck. A microsurgical clip was placedaround the origin of the internal carotid artery (ICA). The distal endof the ECA was ligated with silk and transected. A 6-0 silk was tiedloosely around the ECA stump. The fire-polished tip of a nylon suturewas gently inserted into the ECA stump. The loop of the 6-0 silk wastightened around the stump and the nylon suture was advanced into andthrough the internal carotid artery (ICA), until it rested in theanterior cerebral artery, thereby occluding the anterior communicatingand middle cerebral arteries. After the nylon suture had been in placefor 1 hour, the animal was re-anesthetized, rectal temperature wasrecorded and the suture was removed and the incision closed.

Infarct volume was determined by anesthetizing the animals with anintraperitoneal injection of sodium pentobarbital (50 mg/kg) and thenremoving the brains. The brains were then sectioned into four 2-mmsections through the infracted region and placed in 2%triphenyltetrazolium chloride (TTC) for 30 minutes. After, the sectionswere placed in 4% paraformaldehyde over night. The infarct area in eachsection was determined with a computer-assisted image analysis system,consisting of a Power Macintosh computer equipped with a Quick Captureframe grabber card, Hitachi CCD camera mounted on a camera stand. NIHImage Analysis Software, v. 1.55 was used. The images were captured andthe total area of infarct was determined over the sections. A singleoperator blinded to treatment status performed all measurements. Summingthe infarct volumes of the sections calculated the total infarct volume.The results are expressed as the mean±standard deviation (SD). Thesignificance of difference in the infarct volume data was analyzed usinga t-test.

As illustrated by the data in Table 2, TTP-4000 was more efficaciousthan sRAGE in limiting the area of infarct in these animals suggestingthat TTP-4000, because of its better half-life in plasma, was able tomaintain greater protection in these mice.

Example 6 Detection of RAGE Fusion Protein by ELISA

Initially, 50 uL of the RAGE specific monoclonal antibody 1HB1011 at aconcentration of 10 ug/mL in 1×PBS pH 7.3 is coated on plates viaovernight incubation. When ready for use, plates are washed three timeswith 300 uL of 1× Imidazole-Tween wash buffer and blocked with 1% BSA.The samples (diluted) and standard dilutions of known TTP-4000 dilutionsare added at 100 uL final volume. The samples are allowed to incubate atroom temperature for one hour. After incubation, the plates are platesare washed three times. A Goat Anti-human IgG1 1 (Sigma A3312) APconjugate in 1×PBS with 1% BSA is added and allowed to incubate at roomtemperature for 1 hour. The plates are washed three times. Color waselucidated with paranitrophenylphosphate.

Example 7 Quantification of RAGE Ligand Binding to RAGE Fusion Protein

FIG. 15 shows saturation-binding curves with TTP-4000 to variousimmobilized known RAGE ligands. The ligands are immobilized on amicrotiter plate and incubated in the presence of increasingconcentrations of fusion protein from 0 to 360 nM. The fusionprotein-ligand interaction is detected using a polyclonal antibodyconjugated with alkaline phosphatase that is specific for the IgGportion of the fusion chimera. Relative Kds were calculated usingGraphpad Prizm software and match with established literature values ofRAGE-RAGE ligand values. HMG1B=Ampoterin, CML=Carboxymethyl Lysine, Abeta=Amyloid beta 1-40.

The foregoing is considered as illustrative only of the principal of theinvention. Since numerous modifications and changes will readily occurto those skilled in the art, it is not intended to limit the inventionto the exact embodiments shown and described, and all suitablemodifications and equivalents falling within the scope of the appendedclaims are deemed within the present inventive concept.

1. A fusion protein comprising a RAGE polypeptide directly linked to apolypeptide comprising a C_(H)2 domain of an immunoglobulin or a portionof a C_(H)2 domain of an immunoglobulin.
 2. The fusion protein of claim1, wherein the RAGE polypeptide comprises a RAGE interdomain linkerlinked to a RAGE immunoglobulin domain such that the C-terminal aminoacid of the RAGE immunoglobulin domain is linked to the N-terminal aminoacid of the interdomain linker, and the C-terminal amino acid of theRAGE interdomain linker is directly linked to the N-terminal amino acidof a polypeptide comprising a C_(H)2 domain of an immunoglobulin, or aportion thereof.
 3. The fusion protein of claim 1, wherein the RAGEpolypeptide comprises a ligand binding site.
 4. The fusion protein ofclaim 3, wherein the RAGE ligand binding site comprises SEQ ID NO: 9 ora sequence 90% identical thereto, or SEQ ID NO: 10 or a sequence 90%identical thereto.
 5. The fusion protein of claim 2, wherein the RAGEpolypeptide comprising an interdomain linker linked to a RAGEimmunoglobulin domain comprises a fragment of a full-length RAGEprotein.
 6. The fusion protein of claim 5, further comprising a firstRAGE immunoglobulin domain and a first RAGE interdomain linker linked toa second RAGE immunoglobulin domain and a second RAGE interdomainlinker, such that the N-terminal amino acid of the first interdomainlinker is linked to the C-terminal amino acid of the first RAGEimmunoglobulin domain, the N-terminal amino acid of the second RAGEimmunoglobulin domain is linked to C-terminal amino acid of the firstinterdomain linker, the N-terminal amino acid of the second interdomainlinker is linked to C-terminal amino acid of the second RAGEimmunoglobulin domain, and the C-terminal amino acid of the RAGE secondinterdomain linker is directly linked to the N-terminal amino acid ofthe C_(H)2 immunoglobulin domain or a portion of a C_(H)2 domain of animmunoglobulin.
 7. The fusion protein of claim 6, comprising the aminoacid sequence SEQ ID NO: 33 or SEQ ID NO:
 34. 8. The fusion protein ofclaim 6, comprising the amino acid sequence SEQ ID NO:
 32. 9. The fusionprotein of claim 6, wherein the RAGE interdomain linker directly linkedto the immunoglobulin C_(H)2 domain or a portion thereof comprises SEQID NO: 22 or a sequence 90% identical thereto, or SEQ ID NO: 24, or asequence 90% identical thereto.
 10. The fusion protein of claim 5,comprising a single RAGE immuglobulin domain linked via a RAGEinterdomain linker to the N-terminal amino acid of a polypeptidecomprising a C_(H)2 immunoglobulin domain or a portion of a C_(H)2domain of an immunoglobulin.
 11. The fusion protein of claim 10,comprising the amino acid sequence SEQ ID NO: 36 or SEQ ID NO:
 37. 12.The fusion protein of claim 10, comprising the amino acid sequence SEQID NO:
 35. 13. The fusion protein of claim 10, wherein the RAGE linkerdirectly linked to the immunoglobulin C_(H)2 comprises SEQ ID NO: 21 ora sequence 90% identical thereto or SEQ ID NO: 23 or a sequence 90%identical thereto.
 14. An isolated nucleic acid sequence encoding afusion protein comprising a RAGE polypeptide directly linked apolypeptide comprising a C_(H)2 domain of an immunoglobulin or a portionof a C_(H)2 domain of an immunoglobulin.
 15. The isolated nucleic acidsequence of claim 14, wherein the RAGE polypeptide comprises a RAGEinterdomain linker linked to a RAGE immunoglobulin domain such that theC-terminal amino acid of the RAGE immunoglobulin domain is linked to theN-terminal amino acid of the interdomain linker, and the C-terminalamino acid of the RAGE interdomain linker is directly linked to theN-terminal amino acid of a polypeptide comprising a C_(H)2 domain of animmunoglobulin, or a portion thereof.
 16. The nucleic acid of claim 15,comprising SEQ ID NO:30, or a fragment thereof, or SEQ ID NO: 31, or afragment thereof.
 17. An expression vector that encodes for a fusionprotein comprising a RAGE polypeptide directly linked to a polypeptidecomprising a C_(H)2 domain of an immunoglobulin or a portion of a C_(H)2domain of an immunoglobulin.
 18. The expression vector of claim 17,wherein the RAGE polypeptide comprises a RAGE interdomain linker linkedto a RAGE immunoglobulin domain such that the C-terminal amino acid ofthe RAGE immunoglobulin domain is linked to the N-terminal amino acid ofthe interdomain linker, and the C-terminal amino acid of the RAGEinterdomain linker is directly linked to the N-terminal amino acid of apolypeptide comprising a C_(H)2 domain of an immunoglobulin, or aportion thereof.
 19. The expression vector of claim 17, comprising thenucleic acid sequence SEQ ID NO:30, or a fragment thereof, or SEQ ID NO:31, or a fragment thereof.
 20. A cell transfected with the expressionvector of claim 17, such that the cell expresses a fusion proteincomprising a RAGE polypeptide directly linked to a polypeptidecomprising a C_(H)2 domain of an immunoglobulin or a portion of a C_(H)2domain of an immunoglobulin.
 21. A composition comprising atherapeutically effective amount of a RAGE fusion protein in apharmaceutical carrier, wherein the RAGE fusion protein comprises a RAGEpolypeptide directly linked to a polypeptide comprising a C_(H)2 domainof an immunoglobulin or a portion of a C_(H)2 domain of animmunoglobulin.
 22. The composition of claim 21, wherein the RAGEpolypeptide comprises a RAGE interdomain linker linked to a RAGEimmunoglobulin domain such that the C-terminal amino acid of the RAGEimmunoglobulin domain is linked to the N-terminal amino acid of theinterdomain linker, and the C-terminal amino acid of the RAGEinterdomain linker is directly linked to the N-terminal amino acid of apolypeptide comprising a C_(H)2 domain of an immunoglobulin, or aportion thereof.
 23. The composition of claim 21, wherein the RAGEpolypeptide comprises a ligand binding site.
 24. The composition ofclaim 23, wherein the RAGE ligand binding site comprises SEQ ID NO: 9 ora sequence 90% identical thereto, or SEQ ID NO: 10 or a sequence 90%identical thereto.
 25. The composition of claim 22, wherein the RAGEpolypeptide comprising an interdomain linker linked to a RAGEimmunoglobulin domain comprises a fragment of a full-length RAGEprotein.
 26. The composition of claim 25, wherein the RAGE polypeptidecomprises a first RAGE immunoglobulin domain and a first RAGEinterdomain linker linked to a second RAGE immunoglobulin domain and asecond RAGE interdomain linker, such that the N-terminal amino acid ofthe first interdomain linker is linked to the C-terminal amino acid ofthe first RAGE immunoglobulin domain, the N-terminal amino acid of thesecond RAGE immunoglobulin domain is linked to C-terminal amino acid ofthe first interdomain linker, the N-terminal amino acid of the secondinterdomain linker is linked to C-terminal amino acid of the second RAGEimmunoglobulin domain, and the C-terminal amino acid of the RAGE secondinterdomain linker is directly linked to the N-terminal amino acid ofthe C_(H)2 immunoglobulin domain or a portion of a C_(H)2 domain of animmunoglobulin.
 27. The composition of claim 26, wherein the RAGE fusionprotein comprises the amino acid sequence SEQ ID NO: 33 or SEQ ID NO:34.
 28. The composition of claim 25, wherein the fusion proteincomprises a single RAGE immunoglobulin domain linked via a RAGEinterdomain linker to the N-terminal amino acid of a polypeptidecomprising a C_(H)2 immunoglobulin domain or a portion of a C_(H)2domain of an immunoglobulin.
 29. The composition of claim 28, whereinthe RAGE fusion protein comprises the amino acid sequence SEQ ID NO: 36or SEQ ID NO:
 37. 30. The composition of claim 21, wherein the RAGEfusion protein is formulated as an injectable solution.
 31. Thecomposition of claim 21, wherein the RAGE fusion protein is formulatedas a sterile lyophilized powder.
 32. A method of making a RAGE fusionprotein comprising the step of covalently linking a RAGE polypeptide toa polypeptide comprising a C_(H)2 domain of an immunoglobulin or aportion of a C_(H)2 domain of an immunoglobulin.
 33. The method of claim32, wherein the RAGE polypeptide comprises a RAGE interdomain linkerlinked to a RAGE immunoglobulin domain such that the C-terminal aminoacid of the RAGE immunoglobulin domain is linked to the N-terminal aminoacid of the interdomain linker, and the C-terminal amino acid of theRAGE interdomain linker is directly linked to the N-terminal amino acidof a polypeptide comprising a C_(H)2 domain of an immunoglobulin, or aportion thereof.
 34. The method of claim 32, wherein the RAGEpolypeptide comprises a ligand binding site.
 35. The method of claim 34,wherein the RAGE ligand binding site comprises SEQ ID NO: 9 or asequence 90% identical thereto, or SEQ ID NO: 10 or a sequence 90%identical thereto.
 36. The method of claim 33, wherein the RAGEpolypeptide comprising an interdomain linker linked to a RAGEimmunoglobulin domain comprises a fragment of a full-length RAGEprotein.
 37. The method of claim 36, wherein RAGE polypeptide comprisesa first RAGE immunoglobulin domain and a first RAGE interdomain linkerlinked to a second RAGE immunoglobulin domain and a second RAGEinterdomain linker, such that the N-terminal amino acid of the firstinterdomain linker is linked to the C-terminal amino acid of the firstRAGE immunoglobulin domain, the N-terminal amino acid of the second RAGEimmunoglobulin domain is linked to C-terminal amino acid of the firstinterdomain linker, the N-terminal amino acid of the second interdomainlinker is linked to C-terminal amino acid of the second RAGEimmunoglobulin domain, and the C-terminal amino acid of the RAGE secondinterdomain linker is directly linked to the N-terminal amino acid ofthe C_(H)2 immunoglobulin domain or a portion of a C_(H)2 domain of animmunoglobulin.
 38. The method of claim 36, wherein the RAGE polypeptidecomprises a single RAGE immuglobulin domain linked via a RAGEinterdomain linker to the N-terminal amino acid of a polypeptidecomprising a C_(H)2 immunoglobulin domain or a portion of a C_(H)2domain of an immunoglobulin.
 39. The method of claim 32, wherein thefusion protein is encoded by a recombinant DNA construct.
 40. The methodof claim 32, further comprising the step of incorporating the DNAconstruct into an expression vector.
 41. The method of claim 40, furthercomprising transfecting the expression vector into a host cell.
 42. Amethod of treating a RAGE-mediated disorder in a subject comprisingadministering to a subject a RAGE fusion protein comprising a RAGEpolypeptide directly linked a polypeptide comprising a C_(H)2 domain ofan immunoglobulin or a portion of a C_(H)2 domain of an immunoglobulin.43. The method of claim 42, wherein the RAGE polypeptide comprises aRAGE interdomain linker linked to a RAGE immunoglobulin domain such thatthe C-terminal amino acid of the RAGE immunoglobulin domain is linked tothe N-terminal amino acid of the interdomain linker, and the C-terminalamino acid of the RAGE interdomain linker is directly linked to theN-terminal amino acid of a polypeptide comprising a C_(H)2 domain of animmunoglobulin, or a portion thereof.
 44. The method of claim 42,wherein the RAGE ligand binding site comprises SEQ ID NO: 9 or asequence 90% identical thereto, or SEQ ID NO: 10 or a sequence 90%identical thereto.
 45. The method of claim 43, wherein the RAGEpolypeptide comprising an interdomain linker linked to a RAGEimmunoglobulin domain comprises a fragment of a full-length RAGEprotein.
 46. The method of claim 45, further comprising a first RAGEimmunoglobulin domain and a first RAGE interdomain linker linked to asecond RAGE immunoglobulin domain and a second RAGE interdomain linker,such that the N-terminal amino acid of the first interdomain linker islinked to the C-terminal amino acid of the first RAGE immunoglobulindomain, the N-terminal amino acid of the second RAGE immunoglobulindomain is linked to C-terminal amino acid of the first interdomainlinker, the N-terminal amino acid of the second interdomain linker islinked to C-terminal amino acid of the second RAGE immunoglobulindomain, and the C-terminal amino acid of the RAGE second interdomainlinker is directly linked to the N-terminal amino acid of the C_(H)2immunoglobulin domain or a portion of a C_(H)2 domain of animmunoglobulin.
 47. The method of claim 46, wherein the RAGE fusionprotein comprises the amino acid sequence SEQ ID NO: 33 or SEQ ID NO:34.
 48. The method of claim 46, wherein the RAGE fusion proteincomprises the amino acid sequence SEQ ID NO:
 35. 49. The method of claim46, wherein the RAGE interdomain linker directly linked to theimmunoglobulin C_(H)2 domain or a portion thereof, comprises SEQ ID NO:22 or a sequence 90% identical thereto, or SEQ ID NO: 24 or a sequence90% identical thereto.
 50. The method of claim 45, comprising a singleRAGE immuglobulin domain linked via a RAGE interdomain linker to theN-terminal amino acid of a polypeptide comprising a C_(H)2immunoglobulin domain or a portion of a C_(H)2 domain of animmunoglobulin.
 51. The method of claim 50, wherein the RAGE fusionprotein comprises the amino acid sequence SEQ ID NO: 36 or SEQ ID NO:37.
 52. The method of claim 50, wherein the RAGE fusion proteincomprises the amino acid sequence SEQ ID NO:
 35. 53. The method of claim50, wherein the RAGE interdomain linker directly linked to theimmunoglobulin C_(H)2 or a portion thereof, comprises SEQ ID NO: 21 or asequence 90% identical thereto, or SEQ ID NO: 23 or a sequence 90%identical thereto.
 54. The method of claim 42, comprising intravenousadministration of the RAGE fusion protein to the subject.
 55. The methodof claim 42, comprising intraperitoneal administration of the RAGEfusion protein to the subject.
 56. The method of claim 42, comprisingsubcutaneous administration of the RAGE fusion protein to the subject.57. The method of claim 42, wherein the fusion protein is used to treata symptom of diabetes or a symptom of diabetic late complications. 58.The method of claim 57, wherein the symptom of diabetes or diabetic latecomplications comprises diabetic nephropathy.
 59. The method of claim57, wherein the symptom of diabetes or diabetic late complicationscomprises diabetic retinopathy.
 60. The method of claim 57, wherein thesymptom of diabetes or diabetic late complications comprises a diabeticfoot ulcer.
 61. The method of claim 57, wherein the symptom of diabetesor diabetic late complications comprises a cardiovascular complication.62. The method of claim 57, wherein the symptom of diabetes or diabeticlate complications comprises diabetic neuropathy.
 63. The method ofclaim 42, wherein the fusion protein is used to treat amyloidosis. 64.The method of claim 42, wherein the fusion protein is used to treatAlzheimer's disease.
 65. The method of claim 42, wherein the fusionprotein is used to treat cancer.
 66. The method of claim 42, wherein thefusion protein is used to treat inflammation associated withautoimmunity.
 67. The method of claim 42, wherein the fusion protein isused to treat inflammation associated with inflammatory bowel disease.68. The method of claim 42, wherein the fusion protein is used to treatinflammation associated with rheumatoid arthritis.
 69. The method ofclaim 42, wherein the fusion protein is used to treat inflammationassociated with psoriasis.
 70. The method of claim 42, wherein thefusion protein is used to treat inflammation associated with multiplesclerosis.
 71. The method of claim 42, wherein the fusion protein isused to treat inflammation associated with hypoxia.
 72. The method ofclaim 42, wherein the fusion protein is used to treat inflammationassociated with stroke.
 73. The method of claim 42, wherein the fusionprotein is used to treat inflammation associated with heart attack. 74.The method of claim 42, wherein the fusion protein is used to treatinflammation associated with hemorraghic shock.
 75. The method of claim42, wherein the fusion protein is used to treat inflammation associatedwith sepsis.
 76. The method of claim 42, wherein the fusion protein isused to treat inflammation associated with organ transplantation. 77.The method of claim 42, wherein the fusion protein is used to treatinflammation associated with impaired wound healing.
 78. The method ofclaim 42, wherein the fusion protein is used to treat kidney failure.